Compositions, kits and related methods for the detection and/or monitoring of pseudomonas aeruginosa

ABSTRACT

The present invention provides compositions, methods and kits for the species-specific detection of  pseudomonas aeruginosa.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/909,687, filed Apr. 2, 2007, wherethis provisional application is incorporated herein by reference in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 390082_(—)402_SEQUENCE_LISTING.txt. The textfile is 18 KB, was created on Apr. 2, 2008, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions, methods and kits for thespecies-specific identification of Pseudomonas aeruginosa, which may bepresent either alone or as a component, large or small, of a homogeneousor heterogeneous mixture of nucleic acids in a sample taken for testing,e.g., for diagnostic testing, for screening of blood products, formicrobiological detection in bioprocesses, food, water, industrial orenvironmental samples, and for other purposes.

2. Description of the Related Art

The detection and/or quantitation of specific nucleic acid sequences isan important technique for identifying and classifying microorganisms,diagnosing infectious diseases, detecting and characterizing geneticabnormalities, identifying genetic changes associated with cancer,studying genetic susceptibility to disease, measuring response tovarious types of treatment, and the like. Such procedures are alsouseful in detecting and quantifying microorganisms in foodstuffs, water,industrial and environmental samples, seed stocks, and other types ofmaterial where the presence of specific microorganisms may need to bemonitored.

Nucleic acid amplification assays are well suited for the detection ofmicroorganisms in the context of clinical laboratory testing, bioprocessmonitoring, or any other setting in which the detection of a specificmicroorganisms in a particular sample type is desired, by offering highsensitivity and rapid time-to-result relative to conventionalmicrobiological techniques. In addition, amplification methods can beused in the detection of the vast number of microorganisms that aredifficult or impossible to culture on synthetic media. Nevertheless,there are limitations associated with these approaches, many stemmingfrom the high level of sensitivity of nucleic acid amplification methodsand the resulting amplification of unintended side-products.

Pseudomonas aeruginosa is a gram-negative bacteria that infects humansand can be particularly difficult to treat. In addition, this organismis one of several known contaminant organisms in many biopharmaceuticalprocess streams. Therefore, sensitive and rapid methods for detectingand monitoring the presence of Pseudomonas aeruginosa and other relatedorganisms are continually being sought. While the rapid and accuratedetection and/or quantitation of Pseudomonas aeruginosa is highlydesirable, it has been difficult to achieve in practice usingconventional reagents and techniques. For example, laboratory culturetechniques involve incubating samples for 24-48 hours to allow theorganisms to multiply to macroscopically detectable levels. Subculturetechniques and metabolic assays are then required to distinguishPseudomonas aeruginosa from related pseudomonads and other entericbacteria and may require an additional 24-48 hours.

Accordingly, there remains a need in the art for a rapid and robustdetection system that can specifically and selectively identifyPseudomonas aeruginosa in a sample of interest. As describe furtherherein, the present invention meets these needs and offers other relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

The present invention is drawn generally to compositions, kits andmethods used in the detection of Pseudomonas aeruginosa, which offerimprovements and other advantages in relation to specificity,sensitivity and speed of detection. As discussed further below, theinvention has identified a particular region of the Pseudomonasaeruginosa 23s rRNA as a preferred target for nucleic acid amplificationreactions which provide these improvements and other advantages.

Therefore, according to one aspect of the invention, there are providedcompositions for use in a Pseudomonas aeruginosa transcription-mediatedamplification assay, where the compositions comprise a T7 provideroligonucleotide that targets the complement of a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about725-825 of E. coli 23s rRNA reference sequence (accession no. V00331),and a non-T7 primer oligonucleotide that targets a sequence in a regionof Pseudomonas aeruginosa 23s rRNA corresponding to bases from about845-950 of E. coli 23s rRNA. As further described and establishedherein, use of T7 provider oligonucleotides and non-T7 primeroligonucleotides having particularly defined specificities within thisregion results in improved sensitivity and selectivity intranscription-mediated amplification reactions for the detection ofPseudomonas aeruginosa.

In a particular embodiment of this aspect of the invention, the T7provider targets the complement of a sequence in a region of Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 725-775 of E. coli23s rRNA, and the non-T7 primer targets a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about900-950 of E. coli 23s rRNA.

In a more particular embodiment, the T7 provider targets the complementof a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 739-766 of E. coli 23s rRNA, and thenon-T7 primer targets a sequence in a region of the Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 918-943 of E. coli23s rRNA.

In a more specific embodiment, the T7 provider is selected from SEQ IDNO:2, SEQ ID NO:1, SEQ ID NO:11, or SEQ ID NO:14, and the non-T7 primeris selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:19, or SEQ IDNO:15, as defined herein.

A particularly preferred composition of the invention comprises the T7provider SEQ ID NO:2 and the non-T7 primer oligonucleotide SEQ ID NO:24,as defined herein.

In addition to the T7 provider oligonucleotides and non-T7 primeroligonucleotides discussed above, the compositions according to theinvention can further comprise one or more additional oligonucleotidetypes and/or other amplification reagents that serve to facilitate orimprove one or more aspects of the transcription-mediated amplificationreaction, such as detection oligonucleotides, extend oligonucleotides,blocker oligonucleotides and the like.

For example, in one embodiment, the compositions of the invention willfurther comprise a detection oligonucleotide, preferably a torcholigonucleotide or molecular beacon. In a particular embodiment, thedetection oligonucleotide is a torch oligonucleotide selected from SEQID NO:51, SEQ ID NO:54, SEQ ID NO:50, or SEQ ID NO:56, as definedherein.

The compositions of the invention may also further comprise an extendoligonucleotide. In a particular embodiment, the extend oligonucleotideis selected from SEQ ID NO:43 or SEQ ID NO:44, as defined herein.

The compositions of the invention may also further comprise a blockeroligonucleotide. In a particular embodiment, the blocker oligonucleotideis selected from SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:40, or SEQ IDNO:42, as defined herein.

In one preferred embodiment of the invention, the composition comprisesthe T7 provider oligonucleotide SEQ ID NO:2 and the non-T7 primeroligonucleotide SEQ ID NO:24, and optionally further comprising blockeroligonucleotide SEQ ID NO:29, the torch oligonucleotide SEQ ID NO:54,the extend oligonucleotide SEQ ID NO:44, the target captureoligonucleotide SEQ ID NO:68 and, optionally, the target capture helperoligonucleotide SEQ ID NO:73, as defined herein.

According to another aspect of the invention, there are provided kitsfor performing a Pseudomonas aeruginosa transcription-mediatedamplification assay, where the kits comprise a T7 provideroligonucleotide that targets the complement of a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about725-825 of E. coli 23s rRNA, and a non-T7 primer oligonucleotide thattargets a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 845-950 of E. coli 23s rRNA.

In a particular embodiment of this aspect of the invention, the T7provider targets the complement of a sequence in a region of Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 725-775 of E. coli23s rRNA, and the non-T7 primer targets a sequence in a region of thePseudomonas aeruginosa 23s rRNA corresponding to bases from about900-950 of E. coli 23s rRNA.

In another particular embodiment, the T7 provider targets the complementof a sequence in a region of the Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 739-766 of E. coli 23s rRNA, and thenon-T7 primer targets a sequence in a region of the Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 918-943 of E. coli23s rRNA.

In a more specific embodiment, the T7 provider is selected from SEQ IDNO:2, SEQ ID NO:1, SEQ ID NO:11, or SEQ ID NO:14, and the non-T7 primeris selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:19, or SEQ IDNO:15.

In another specific embodiment, the T7 provider is SEQ ID NO:2 and thenon-T7 primer is SEQ ID NO:24.

The kits of the invention may further comprise, in addition to the T7provider oligonucleotide and non-T7 primer oligonucleotide, one of moreadditional oligonucleotides and/or other reagents that are desired orpreferred in a transcription-mediated amplification reaction, such asdetection oligonucleotides, extend oligonucleotides, blockeroligonucleotides and the like. For example, a kit of the invention mayfurther comprise a detection oligonucleotide, such as a torcholigonucleotide or molecular beacon. In a particular embodiment, forexample, the kit comprises a torch oligonucleotide that is selected fromSEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:50, or SEQ ID NO:56.

A kit of the invention may also further comprise an extendoligonucleotide. In a particular embodiment, the extend oligonucleotideis selected from SEQ ID NO:43 or SEQ ID NO:44.

A kit of the invention may also further comprise a blockeroligonucleotide. In a particular embodiment, the blocker oligonucleotideis selected from SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:40, or SEQ IDNO:42.

In a more specific embodiment, a kit of the invention comprises the T7provider oligonucleotide, SEQ ID NO:2 and the non-T7 primeroligonucleotide, SEQ ID NO:24, and optionally further comprising blockeroligonucleotide SEQ ID NO:29, the torch oligonucleotide SEQ ID NO:54,the extend oligonucleotide SEQ ID NO:44, the target captureoligonucleotide SEQ ID NO:69 and, optionally, the target capture helperoligonucleotide SEQ ID NO:73.

According to yet another aspect of the invention, there are providedmethods for detecting the presence of Pseudomonas aeruginosa in asample, wherein the method involves performing a transcription-mediatedamplification assay using a T7 provider oligonucleotide and a non-T7primer oligonucleotide, wherein the T7 provider oligonucleotide targetsthe complement of a sequence in a region of Pseudomonas aeruginosa 23srRNA corresponding to bases from about 725-825 of E. coli 23s rRNA, andthe non-T7 primer oligonucleotide targets a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about845-950 of E. coli 23s rRNA.

In a particular embodiment, the T7 provider targets the complement of asequence in a region of Pseudomonas aeruginosa 23s rRNA corresponding tobases from about 725-775 of E. coli 23s rRNA, and the non-T7 primertargets a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 900-950 of E. coli 23s rRNA.

In another particular embodiment, the T7 provider targets the complementof a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 739-766 of E. coli 23s rRNA, and thenon-T7 primer targets a sequence of a region of the Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 918-943 of E. coli23s rRNA.

In a more specific embodiment, the T7 provider is selected from SEQ IDNO:2, SEQ ID NO:1, SEQ ID NO:11 or SEQ ID NO:14, and the non-T7 primeris selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:19 or SEQ IDNO:15.

In another more specific embodiment, the T7 provider is SEQ ID NO:2 andthe non-T7 primer is SEQ ID NO:24.

As with the compositions and kits discussed above, the methods accordingto this aspect of the invention may further comprise additionalancillary oligonucleotides and/or reagents effective in atranscription-mediated amplification reaction, such as a detectionoligonucleotide, blocker oligonucleotide, extend oligonucleotide, andthe like.

For example, the methods may employ the use of a detectionoligonucleotide, such as a torch oligonucleotide or molecular beacon. Inone embodiment, the torch oligonucleotide is selected from SEQ ID NO:51,SEQ ID NO:54, SEQ ID NO:50 or SEQ ID NO:56.

The methods may also employ the use of an extend oligonucleotide, suchas SEQ ID NO:43 or SEQ ID NO:44, or a blocker oligonucleotide such asSEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:40 or SEQ ID NO:42.

In a more specific embodiment of this aspect of the invention, thetranscription-mediated amplification method employs the use of the T7provider oligonucleotide SEQ ID NO:2 and the non-T7 primeroligonucleotide SEQ ID NO:24, and further employ the use of the blockeroligonucleotide SEQ ID NO:29, the torch oligonucleotide SEQ ID NO:54,the extend oligonucleotide SEQ ID NO:44, the target captureoligonucleotide SEQ ID NO:69 and, optionally, the target capture helperoligonucleotide, SEQ ID NO:73.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a real-time fluorescence signal that was obtained for anamplification of approximately 10 CFU of P. aeruginosa compared to thatwhich was obtained for 10⁵ CFU of closely related pseudomonads.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to compositions, methods andkits for detecting, monitoring and/or quantitating the presence ofPseudomonas aeruginosa in samples, such as clinical samples, bioprocesssamples, food samples, water samples, industrial samples, environmentalsamples, or any other sample type known or suspected of containingPseudomonas aeruginosa. Specific compositions, methods and kits of thepresent invention provide improved sensitivity, specificity andselectivity in the amplification-based detection of Pseudomonasaeruginosa.

As a result of extensive analyses of amplification oligonucleotidesspecific for Pseudomonas aeruginosa, the present invention hasidentified a particular region of Pseudomonas aeruginosa, correspondingto the region of E. coli 23s rRNA reference sequence (accession no.V00331) from about 700 to 1000 nucleotide bases (hereinafter referred toas the “800 region”), as a preferred target for amplification-baseddetection of Pseudomonas aeruginosa. Accordingly, the present inventionrelates to amplification oligonucleotides, compositions, reactionsmixtures, kits, and the like, as well as their use in thespecies-specific detection of Pseudomonas aeruginosa in a sample ofinterest.

The terms and concepts of the invention have meanings as set forthherein unless expressly stated to the contrary and/or unless contextspecifically dictates otherwise. Unless defined otherwise, scientificand technical terms used herein have the same meaning as commonlyunderstood by those skilled in the relevant art. General definitions maybe found in technical books relevant to the art of molecular biology,e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.) or TheHarper Collins Dictionary of Biology (Hale & Marham, 1991, HarperPerennial, New York, N.Y.). Unless mentioned otherwise, techniquesemployed or contemplated herein are standard methodologies well known toone of ordinary skill in the art. The examples included hereinillustrate some preferred embodiments.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.Further, each reference cited herein is specifically incorporated hereinby reference in its entirety.

Nucleic Acid Amplification and Detection

It will be understood by the skilled artisan that the preferredoligonucleotides, compositions, reaction mixtures and kits of thepresent invention are used in nucleic acid amplification methods for theimproved detection of Pseudomonas aeruginosa. While such methods andtechniques are well known and established, illustrative and preferredaspects of nucleic acid amplification, detection, etc., is discussedbelow.

Nucleic Acids

The term “nucleic acid” is intended to encompass a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, virus genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid. The term “polynucleotide” may beused herein to denote a nucleic acid chain. Throughout this application,nucleic acids are designated by the 5′-terminus to the 3′-terminus.Standard nucleic acids, e.g., DNA and RNA, are typically synthesized“3′-to-5′,” i.e., by the addition of nucleotides to the 5′-terminus of agrowing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me). As used herein, methoxyoligonucleotides containing “T” residues have a methoxy group at the 2′position of the ribose moiety, and a uracil at the base position of thenucleotide.

A “non-nucleotide unit” is a unit which does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

Target Nucleic Acid/Target Sequence

A “target nucleic acid” is a nucleic acid comprising a “target sequence”to be amplified. Target nucleic acids may be DNA or RNA as describedherein, and may be either single-stranded or double-stranded. The targetnucleic acid may include other sequences besides the target sequencewhich may not be amplified. Typical target nucleic acids include virusgenomes, bacterial genomes, fungal genomes, plant genomes, animalgenomes, rRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells,mitochondrial DNA, or chromosomal DNA.

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinicalspecimens, e.g., blood, urine, saliva, feces, semen, or spinal fluid,from criminal evidence, from environmental samples, e.g., water or soilsamples, from food, from industrial samples, from cDNA libraries, orfrom total cellular RNA. By “isolated” it is meant that a samplecontaining a target nucleic acid is taken from its natural milieu, butthe term does not connote any degree of purification. If necessary,target nucleic acids of the present invention are made available forinteraction with the various oligonucleotides of the present invention.This may include, for example, cell lysis or cell permeabilization torelease the target nucleic acid from cells, which then may be followedby one or more purification steps, such as a series of isolation andwash steps. See, e.g., Clark et al., “Method for Extracting NucleicAcids from a Wide Range of Organisms,” U.S. Pat. No. 5,786,208, thecontents of which are hereby incorporated by reference herein. This isparticularly important where the sample may contain components that caninterfere with the amplification reaction, such as, for example, hemepresent in a blood sample. See Ryder et al., “Amplification of NucleicAcids from Mononuclear Cells Using Iron Complexing and Other Agents,”U.S. Pat. No. 5,639,599, the contents of which are hereby incorporatedby reference herein. Methods to prepare target nucleic acids fromvarious sources for amplification are well known to those of ordinaryskill in the art. Target nucleic acids of the present invention may bepurified to some degree prior to the amplification reactions describedherein, but in other cases, the sample is added to the amplificationreaction without any further manipulations.

The term “target sequence” refers to the particular nucleotide sequenceof the target nucleic acid which is to be amplified. The “targetsequence” includes the complexing sequences to which oligonucleotides(e.g., priming oligonucleotides and/or promoter oligonucleotides)complex during the processes of the present invention. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the “target sequence”as present in the target nucleic acid. Where the “target nucleic acid”is originally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a “unique” sequence should bechosen so as to distinguish between unrelated or closely related targetnucleic acids. As will be understood by those of ordinary skill in theart, “unique” sequences are judged from the testing environment. Atleast the sequences recognized by the detection probe (as described inmore detail elsewhere herein) should be unique in the environment beingtested, but need not be unique within the universe of all possiblesequences. Furthermore, even though the target sequence should contain a“unique” sequence for recognition by a detection probe, it is not alwaysthe case that the priming oligonucleotide and/or promoteroligonucleotide are recognizing “unique” sequences. In some embodiments,it may be desirable to choose a target sequence which is common to afamily of related organisms, for example, a sequence which is common toone or more pseudomonads that might be in a sample. In other situations,a very highly specific target sequence, or a target sequence having atleast a highly specific region recognized by the detection probe andamplification oligonucleotides, would be chosen so as to distinguishbetween closely related organisms, for example, between pathogenic andnon-pathogenic E. coli. A target sequence of the present invention maybe of any practical length. A minimal target sequence includes theregion which hybridizes to the priming oligonucleotide (or thecomplement thereof), the region which hybridizes to the hybridizingregion of the promoter oligonucleotide (or the complement thereof), anda region used for detection, e.g., a region which hybridizes to adetection probe, described in more detail elsewhere herein. The regionwhich hybridizes with the detection probe may overlap with or becontained within the region which hybridizes with the primingoligonucleotide (or its complement) or the hybridizing region of thepromoter oligonucleotide (or its complement). In addition to the minimalrequirements, the optimal length of a target sequence depends on anumber of considerations, for example, the amount of secondarystructure, or self-hybridizing regions in the sequence. Typically,target sequences of the present invention range from about 100nucleotides in length to from about 150 to about 250 nucleotides inlength. The optimal or preferred length may vary under differentconditions which can be determined according to the methods describedherein. The term “amplicon” refers to the nucleic acid moleculegenerated during an amplification procedure that is complementary orhomologous to a sequence contained within the target sequence.

Nucleic Acid “Identity”

In certain embodiments, a nucleic acid of the present inventioncomprises a contiguous base region that is at least 80%, 90%, or 100%identical to a contiguous base region of a reference nucleic acid. Forshort nucleic acids, e.g., certain oligonucleotides of the presentinvention, the degree of identity between a base region of a “query”nucleic acid and a base region of a reference nucleic acid can bedetermined by manual alignment. “Identity” is determined by comparingjust the sequence of nitrogenous bases, irrespective of the sugar andbackbone regions of the nucleic acids being compared. Thus, thequery:reference base sequence alignment may be DNA:DNA, RNA:RNA,DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNAand DNA base sequences can be compared by converting U's (in RNA) to T's(in DNA).

Oligonucleotides & Primers

As used herein, the term “oligonucleotide” or “oligo” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods of the present invention, as wellas subsequent detection methods. The oligonucleotide may be DNA and/orRNA and/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent, rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., it may function as a primer if it isspecific for and capable of hybridizing to a complementary strand andcan further be extended in the presence of a nucleic acid polymerase, itmay provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 provider), and itmay function to prevent hybridization or impede primer extension ifappropriately situated and/or modified. Specific oligonucleotides of thepresent invention are described in more detail below.

As used herein, an oligonucleotide can be virtually any length, limitedonly by its specific function in the amplification reaction or indetecting an amplification product of the amplification reaction.However, in certain embodiments, preferred oligonucleotides will containat least about 10, 12, 14, 16, 18 or 20 contiguous bases that arecomplementary to a region of the target nucleic acid sequence or itscomplementary strand. The contiguous bases are preferably at least about80%, more preferably at least about 90%, and most preferably completelycomplementary to the target sequence to which the oligonucleotide binds.Certain preferred oligonucleotides are of lengths generally betweenabout 10-100, 10-75, 10-50 or 10-25 bases long and optionally caninclude modified nucleotides.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Modifications include basemodifications, sugar modifications or backbone modifications. Basemodifications include, but are not limited to the use of the followingbases in addition to adenine, cytidine, guanosine, thymine and uracil:C-5 propyne, 2-amino adenine, 5-methyl cytidine, inosine, and dP and dKbases. The sugar groups of the nucleoside subunits may be ribose,deoxyribose and analogs thereof, including, for example, ribonucleosideshaving a 2′-O-methyl substitution to the ribofuranosyl moiety. SeeBecker et al., U.S. Pat. No. 6,130,038. Other sugar modificationsinclude, but are not limited to 2′-amino, 2′-fluoro,(L)-alpha-threofuranosyl, and pentopyranosyl modifications. Thenucleoside subunits may by joined by linkages such as phosphodiesterlinkages, modified linkages or by non-nucleotide moieties which do notprevent hybridization of the oligonucleotide to its complementary targetnucleic acid sequence. Modified linkages include those linkages in whicha standard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. DNA analogs having a pseudo peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA” and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082. Otherlinkage modifications include, but are not limited to, morpholino bonds.

Non-limiting examples of oligonucleotides or oligos contemplated by thepresent invention include nucleic acid analogs containing bicyclic andtricyclic nucleoside and nucleotide analogs (LNAs). See Imanishi et al.,U.S. Pat. No. 6,268,490; and Wengel et al., U.S. Pat. No. 6,670,461.)Any nucleic acid analog is contemplated by the present inventionprovided the modified oligonucleotide can perform its intended function,e.g., hybridize to a target nucleic acid under stringent hybridizationconditions or amplification conditions, or interact with a DNA or RNApolymerase, thereby initiating extension or transcription. In the caseof detection probes, the modified oligonucleotides must also be capableof preferentially hybridizing to the target nucleic acid under stringenthybridization conditions.

While design and sequence of oligonucleotides for the present inventiondepend on their function as described below, several variables mustgenerally be taken into account. Among the most critical are: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well known aspect ofoligonucleotide design, and various computer programs are readilyavailable to initially screen large numbers of potentialoligonucleotides.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “primer.”

As used in this disclosure, an oligonucleotide having a nucleic acidsequence “comprising” or “consisting of” or “consisting essentially of”a sequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

An oligonucleotide substantially corresponding to a specified nucleicacid sequence means that the referred to oligonucleotide is sufficientlysimilar to the reference nucleic acid sequence such that theoligonucleotide has similar hybridization properties to the referencenucleic acid sequence in that it would hybridize with the same targetnucleic acid sequence under stringent hybridization conditions.

One skilled in the art will understand that substantially correspondingoligonucleotides of the invention can vary from the referred to sequenceand still hybridize to the same target nucleic acid sequence. Thisvariation from the nucleic acid may be stated in terms of a percentageof identical bases within the sequence or the percentage of perfectlycomplementary bases between the probe or primer and its target sequence.Thus, an oligonucleotide of the present invention substantiallycorresponds to a reference nucleic acid sequence if these percentages ofbase identity or complementarity are from 100% to about 80%. Inpreferred embodiments, the percentage is from 100% to about 85%. In morepreferred embodiments, this percentage can be from 100% to about 90%; inother preferred embodiments, this percentage is from 100% to about 95%.One skilled in the art will understand the various modifications to thehybridization conditions that might be required at various percentagesof complementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

A “helper oligonucleotide” or “helper” refers to an oligonucleotidedesigned to bind to a target nucleic acid and impose a differentsecondary and/or tertiary structure on the target to increase the rateand extent of hybridization of a detection probe or otheroligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557, the contents of which areincorporated by reference herein. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides.

Blocking Moiety

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. A blockingmoiety may be a small molecule, e.g., a phosphate or ammonium group, orit may be a modified nucleotide, e.g., a 3′2′ dideoxynucleotide or 3′deoxyadenosine 5′-triphosphate (cordycepin), or other modifiednucleotide. Additional blocking moieties include, for example, the useof a nucleotide or a short nucleotide sequence having a 3′-to-5′orientation, so that there is no free hydroxyl group at the 3′-terminus,the use of a 3′ alkyl group, a 3′ non-nucleotide moiety (see, e.g.,Arnold et al., “Non-Nucleotide Linking Reagents for Nucleotide Probes,”U.S. Pat. No. 6,031,091, the contents of which are hereby incorporatedby reference herein), phosphorothioate, alkane-diol residues, peptidenucleic acid (PNA), nucleotide residues lacking a 3′ hydroxyl group atthe 3′-terminus, or a nucleic acid binding protein. Preferably, the3′-blocking moiety comprises a nucleotide or a nucleotide sequencehaving a 3′-to-5′ orientation or a 3′ non-nucleotide moiety, and not a3′2′-dideoxynucleotide or a 3′ terminus having a free hydroxyl group.Additional methods to prepare 3′-blocking oligonucleotides are wellknown to those of ordinary skill in the art.

Priming Oligonucleotide or Primer

A priming oligonucleotide or “primer” is an oligonucleotide, at leastthe 3′-end of which is complementary to a nucleic acid template, andwhich complexes (by hydrogen bonding or hybridization) with the templateto give a primer:template complex suitable for initiation of synthesisby an RNA- or DNA-dependent DNA polymerase. A priming oligonucleotide isextended by the addition of covalently bonded nucleotide bases to its3′-terminus, which bases are complementary to the template. The resultis a primer extension product. A priming oligonucleotide of the presentinvention is typically at least 10 nucleotides in length, and may extendup to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Suitableand preferred priming oligonucleotides are described herein. Virtuallyall DNA polymerases (including reverse transcriptases) that are knownrequire complexing of an oligonucleotide to a single-stranded template(“priming”) to initiate DNA synthesis, whereas RNA replication andtranscription (copying of RNA from DNA) generally do not require aprimer. By its very nature of being extended by a DNA polymerase, apriming oligonucleotide does not comprise a 3′-blocking moiety.

Promoter Oligonucleotide/Promoter Sequence

As is well known in the art, a “promoter” is a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site. For binding, it was generallythought that such transcriptases required DNA which had been rendereddouble-stranded in the region comprising the promoter sequence via anextension reaction, however, the present inventors have determined thatefficient transcription of RNA can take place even under conditionswhere a double-stranded promoter is not formed through an extensionreaction with the template nucleic acid. The template nucleic acid (thesequence to be transcribed) need not be double-stranded. IndividualDNA-dependent RNA polymerases recognize a variety of different promotersequences, which can vary markedly in their efficiency in promotingtranscription. When an RNA polymerase binds to a promoter sequence toinitiate transcription, that promoter sequence is not part of thesequence transcribed. Thus, the RNA transcripts produced thereby willnot include that sequence.

As used herein, a “promoter oligonucleotide” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter oligonucleotide of thepresent invention comprises a base sequence which hybridizes to a DNAtemplate, where the hybridizing sequence is situated 3′, but notnecessarily adjacent to, a promoter region. The hybridizing portion of apromoter oligonucleotide of the present invention is typically at least10 nucleotides in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. The “second region” comprises apromoter sequence for an RNA polymerase. A promoter oligonucleotide ofthe present invention is engineered so that it is incapable of beingextended by an RNA- or DNA-dependent DNA polymerase, e.g., reversetranscriptase, preferably comprising a blocking moiety at its3′-terminus as described above. Suitable and preferred promoteroligonucleotides are described herein.

Terminating Oligonucleotide

In the present invention, a “terminating oligonucleotide” or “blockeroligo” is an oligonucleotide comprising a base sequence that iscomplementary to a region of the target nucleic acid in the vicinity ofthe 5′-end of the target sequence, so as to “terminate” primer extensionof a nascent nucleic acid that includes a priming oligonucleotide,thereby providing a defined 3′-end for the nascent nucleic acid strand.A terminating oligonucleotide is designed to hybridize to the targetnucleic acid at a position sufficient to achieve the desired 3′-end forthe nascent nucleic acid strand. The positioning of the terminatingoligonucleotide is flexible depending upon its design. A terminatingoligonucleotide may be modified or unmodified. In certain embodiments,terminating oligonucleotides are synthesized with at least one or more2′-O-methyl ribonucleotides. These modified nucleotides havedemonstrated higher thermal stability of complementary duplexes. The2′-O-methyl ribonucleotides also function to increase the resistance ofoligonucleotides to exonucleases, thereby increasing the half-life ofthe modified oligonucleotides. See, e.g., Majlessi et al. (1988) NucleicAcids Res. 26, 2224-9, the contents of which are hereby incorporated byreference herein. Other modifications as described elsewhere herein maybe utilized in addition to or in place of 2′-O-methyl ribonucleotides.For example, a terminating oligonucleotide may comprise PNA or an LNA.See, e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53, thecontents of which are hereby incorporated by reference herein. Aterminating oligonucleotide of the present invention typically includesa blocking moiety at its 3′-terminus to prevent extension. A terminatingoligonucleotide may also comprise a protein or peptide joined to theoligonucleotide so as to terminate further extension of a nascentnucleic acid chain by a polymerase. A terminating oligonucleotide of thepresent invention is typically at least 10 bases in length, and mayextend up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length.Suitable and preferred terminating oligonucleotides are describedherein. It should be noted that while a terminating oligonucleotidetypically or necessarily includes a 3′-blocking moiety, “3′-blocked”oligonucleotides are not necessarily terminating oligonucleotides. Otheroligonucleotides of the present invention, e.g., promoteroligonucleotides and capping oligonucleotides are typically ornecessarily 3′-blocked as well.

Extender Oligonucleotide

An “extender oligonucleotide” or “extend oligo” refers to anoligonucleotide that is the same sense as the T7 provider and may act asa helper oligonucleotide that opens up structure or improvesspecificity. An extender oligonucleotide hybridizes to a DNA templateadjacent to or near the 3′-end of the first region of a promoteroligonucleotide. An extender oligonucleotide preferably hybridizes to aDNA template such that the 5′-terminal base of the extenderoligonucleotide is within 3, 2 or 1 bases of the 3′-terminal base of apromoter oligonucleotide. Most preferably, the 5′-terminal base of anextender oligonucleotide is adjacent to the 3′-terminal base of apromoter oligonucleotide when the extender oligonucleotide and thepromoter oligonucleotide are hybridized to a DNA template. To preventextension of an extender oligonucleotide, a 3′-terminal blocking moietyis typically included. An extender oligonucleotide is preferably 10 to50 nucleotides in length, more preferably 20 to 40 nucleotides inlength, and most preferably 30 to 35 nucleotides in length {seeUS2006/0046265}.

Probe

By “probe” or “detection probe” is meant a molecule comprising anoligonucleotide having a base sequence partly or completelycomplementary to a region of a target sequence sought to be detected, soas to hybridize thereto under stringent hybridization conditions. Aswould be understood by someone having ordinary skill in the art, a probecomprises an isolated nucleic acid molecule, or an analog thereof, in aform not found in nature without human intervention (e.g., recombinedwith foreign nucleic acid, isolated, or purified to some extent).

The probes of this invention may have additional nucleosides ornucleobases outside of the targeted region so long as such nucleosidesor nucleobases do not substantially affect hybridization under stringenthybridization conditions and, in the case of detection probes, do notprevent preferential hybridization to the target nucleic acid. Anon-complementary sequence may also be included, such as a targetcapture sequence (generally a homopolymer tract, such as a poly-A,poly-T or poly-U tail), promoter sequence, a binding site for RNAtranscription, a restriction endonuclease recognition site, or maycontain sequences which will confer a desired secondary or tertiarystructure, such as a catalytic active site or a hairpin structure on theprobe, on the target nucleic acid, or both.

The probes preferably include at least one detectable label. The labelmay be any suitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester. Certain probes of the present invention do not includea label. For example, non-labeled “capture” probes may be used to enrichfor target sequences or replicates thereof, which may then be detectedby a second “detection” probe. See, e.g., Weisburg et al., “Two-StepHybridization and Capture of a Polynucleotide,” U.S. Pat. No. 6,534,273,which is hereby incorporated by reference herein. While detection probesare typically labeled, certain detection technologies do not requirethat the probe be labeled. See, e.g., Nygren et al., “Devices andMethods for Optical Detection of Nucleic Acid Hybridization,” U.S. Pat.No. 6,060,237.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5° C. below the melting temperature of the nucleicacid duplex, and even more preferably at least 10° C. below the meltingtemperature of the reaction mixture.

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, probes of the present inventionhybridize to their target sequences, or replicates thereof, to formstable probe:target hybrids, while at the same time formation of stableprobe:non-target hybrids is minimized. Thus, a probe hybridizes to atarget sequence or replicate thereof to a sufficiently greater extentthan to a non-target sequence, to enable one having ordinary skill inthe art to accurately quantitate the RNA replicates or complementary DNA(cDNA) of the target sequence formed during the amplification.

Probes of a defined sequence may be produced by techniques known tothose of ordinary skill in the art, such as by chemical synthesis, andby in vitro or in vivo expression from recombinant nucleic acidmolecules. Preferably probes are 10 to 100 nucleotides in length, morepreferably 12 to 50 bases in length, and even more preferably 18 to 35bases in length.

Hybridize/Hybridization

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof. The two constituentstrands of this double-stranded structure, sometimes called a hybrid,are held together by hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can also form between baseswhich are not members of these “canonical” pairs. Non-canonical basepairing is well-known in the art. (See, e.g., Roger L. P. Adams et al.,“The Biochemistry Of The Nucleic Acids” (11^(th) ed. 1992).)

“Stringent” hybridization assay conditions refer to conditions wherein aspecific detection probe is able to hybridize with target nucleic acidsover other nucleic acids present in the test sample. It will beappreciated that these conditions may vary depending upon factorsincluding the GC content and length of the probe, the hybridizationtemperature, the composition of the hybridization reagent or solution,and the degree of hybridization specificity sought. Specific stringenthybridization conditions are provided in the disclosure below.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. When a contiguous sequence of nucleotides ofone single-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “perfectly”complementary.

By “preferentially” hybridize is meant that under stringenthybridization assay conditions, certain complementary nucleotides ornucleobase sequences hybridize to form a stable hybrid preferentiallyover other, less stable duplexes.

Nucleic Acid Amplification

As noted, the present invention relates generally to compositions andmethods for detection of Pseudomonas aeruginosa in a sample of interestusing nucleic acid amplification methods. By “amplification” or “nucleicacid amplification” is meant production of multiple copies of a targetnucleic acid that contains at least a portion of the intended specifictarget nucleic acid sequence, as further described herein. The multiplecopies may be referred to as amplicons or amplification products

The compositions and methods of the invention may be performed onessentially any sample type of interest that is known or suspected ofcontaining Pseudomonas aeruginosa. These may include biological samples,clinical samples, industrial samples, and the like. In one preferredembodiment, the sample is a biopharmaceutical process (bioprocess)stream where Pseudomonas aeruginosa is a known or suspected contaminant.A “bioprocess,” as used herein, refers generally to any process in whichliving cells or organisms, or components thereof, are present, eitherintended or unintended. For example, essentially any manufacturing orother process that employs one or more samples or sample streams, atleast one of which contains living cells, organisms, or componentsthereof, or contains such cells, organisms or components as a result ofunintended contamination, is considered a bioprocess. In many suchprocesses it is desirable to have the ability to detect, identify and/orcontrol the presence and/or sources of living cells, organisms orcomponents thereof within a process. Using the methods of the presentinvention, for example, the presence and/or sources of Pseudomonasaeruginosa in one or more bioprocess samples and/or streams may bemonitored in a rapid and sensitive fashion.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.The ligase chain reaction (Weiss, R. 1991, Science 254: 1292), commonlyreferred to as LCR, uses two sets of complementary DNA oligonucleotidesthat hybridize to adjacent regions of the target nucleic acid. The DNAoligonucleotides are covalently linked by a DNA ligase in repeatedcycles of thermal denaturation, hybridization and ligation to produce adetectable double-stranded ligated oligonucleotide product. Anothermethod is strand displacement amplification (Walker, G. et al., 1992,Proc. Natl. Acad. Sci. USA 89:392-396; U.S. Pat. Nos. 5,270,184 and5,455,166), commonly referred to as SDA, which uses cycles of annealingpairs of primer sequences to opposite strands of a target sequence,primer extension in the presence of a dNTPαS to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (European Pat. No. 0 684 315). Otheramplification methods include: nucleic acid sequence based amplification(U.S. Pat. No. 5,130,238), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi, P. etal., 1988, BioTechnol. 6: 1197-1202), commonly referred to as Q-βreplicase; a transcription based amplification method (Kwoh, D. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177); self-sustained sequencereplication (Guatelli, J. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878); and, transcription mediated amplification (U.S. Pat. Nos.5,480,784 and 5,399,491), commonly referred to as TMA. For furtherdiscussion of known amplification methods see Persing, David H., 1993,“In Vitro Nucleic Acid Amplification Techniques” in Diagnostic MedicalMicrobiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, D.C.).

In a preferred embodiment of the invention, Pseudomonas aeruginosa isdetected by a transcription-based amplification technique. One preferredtranscription-based amplification system is transcription-mediatedamplification (TMA), which employs an RNA polymerase to produce multipleRNA transcripts of a target region. Exemplary TMA amplification methodsare described U.S. Pat. Nos. 5,480,784, 5,399,491, US 2006/0046265, andreferences cited therein, the contents of which are incorporated hereinby reference in their entireties. TMA uses a “promoter-primer” thathybridizes to a target nucleic acid in the presence of a reversetranscriptase and an RNA polymerase to form a double-stranded promoterfrom which the RNA polymerase produces RNA transcripts. Thesetranscripts can become templates for further rounds of TMA in thepresence of a second primer capable of hybridizing to the RNAtranscripts. Unlike PCR, LCR or other methods that require heatdenaturation, TMA is an isothermal method that uses an RNase H activityto digest the RNA strand of an RNA:DNA hybrid, thereby making the DNAstrand available for hybridization with a primer or promoter-primer.Generally, the RNase H activity associated with the reversetranscriptase provided for amplification is used.

In one version of the TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce promoter-primer extension. From anunmodified promoter-primer, reverse transcriptase creates a cDNA copy ofthe target RNA, while RNase H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer”. From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNase H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

Another version of TMA uses one primer and one or more additionalamplification oligomers to amplify nucleic acids in vitro, makingtranscripts (amplicons) that indicate the presence of the targetsequence in a sample (previously described in detail in Becker et al.,US Pub. No. 2006/0046265, the details of which are hereby incorporatedby reference herein). Briefly, the single-primer TMA method uses aprimer (or “priming oligomer”), a modified promoter oligomer (or“promoter-provider”) that is modified to prevent the initiation of DNAsynthesis from its 3′ end (e.g., by including a 3′-blocking moiety) and,optionally, a binding molecule (e.g., a 3′-blocked extender oligomer) toterminate elongation of a cDNA from the target strand. This methodsynthesizes multiple copies of a target sequence and includes the stepsof treating a target RNA that contains a target sequence with a primingoligomer and a binding molecule, where the primer hybridizes to the 3′end of the target strand. RT initiates primer extension from the 3′ endof the primer to produce a cDNA which is in a duplex with the targetstrand (e.g., RNA:cDNA). When a binding molecule, such as a 3′ blockedextender oligomer, is used in the reaction, it binds to the targetnucleic acid adjacent near the 5′ end of the target sequence. That is,the binding molecule binds to the target strand next to the 5′ end ofthe target sequence to be amplified. When the primer is extended by DNApolymerase activity of RT to produce cDNA, the 3′ end of the cDNA isdetermined by the position of the binding molecule becausepolymerization stops when the primer extension product reaches thebinding molecule bound to the target strand. Thus, the 3′ end of thecDNA is complementary to the 5′ end of the target sequence. The RNA:cDNAduplex is separated when RNase (e.g., RNase H of RT) degrades the RNAstrand, although those skilled in the art will appreciate that any formof strand separation may be used. Then, the promoter-provider oligomerhybridizes to the cDNA near the 3′ end of the cDNA strand. Thepromoter-provider oligomer includes a 5′ promoter sequence for an RNApolymerase and a 3′ region complementary to a sequence in the 3′ regionof the cDNA. The promoter-provider oligomer also has a modified 3′ endthat includes a blocking moiety that prevents initiation of DNAsynthesis from the 3′ end of the promoter-provider oligomer. In thepromoter-provide:cDNA duplex, the 3′-end of the cDNA is extended by DNApolymerase activity of RT using the promoter oligomer as a template toadd a promoter sequence to the cDNA and create a functionaldouble-stranded promoter. An RNA polymerase specific for the promotersequence then binds to the functional promoter and transcribes multipleRNA transcripts complementary to the cDNA and substantially identical tothe target region sequence that was amplified from the initial targetstrand. The resulting amplified RNA can then cycle through the processagain by binding the primer and serving as a template for further cDNAproduction, ultimately producing many amplicons from the initial targetnucleic acid present in the sample. Some embodiments of thesingle-primer transcription associated amplification method do notinclude the binding molecule and, therefore, the cDNA product made fromthe primer has an indeterminate 3′ end, but the amplification stepsproceed substantially as described above for all other steps.

Suitable amplification conditions can be readily determined by a skilledartisan in view of the present disclosure. “Amplification conditions”refer to conditions which permit nucleic acid amplification according tothe present invention. Amplification conditions may, in someembodiments, be less stringent than “stringent hybridization conditions”as described herein. Oligos used in the amplification reactions of thepresent invention are specific for and hybridize to their intendedtargets under amplification conditions, but may or may not hybridizeunder more stringent hybridization conditions. On the other hand,detection probes of the present invention hybridize under stringenthybridization conditions. While the Examples section infra providespreferred amplification conditions for amplifying target nucleic acidsequences according to the present invention, other acceptableconditions to carry out nucleic acid amplifications according to thepresent invention could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

The amplification methods of the invention, in certain embodiments, alsopreferably employ the use of one or more other types of oligos that areeffective for improving the sensitivity, selectivity, efficiency, etc.,of the amplification reaction. These may include, for example,terminating oligonucleotides, extender or helper oligonucleotides, andthe like.

Target Capture

In certain embodiments, it may be preferred to purify or enrich a targetnucleic acid from a sample prior to amplification, for example using atarget capture approach. “Target capture” (TC) refers generally tocapturing a target polynucleotide onto a solid support, such asmagnetically attractable particles, wherein the solid support retainsthe target polynucleotide during one or more washing steps of the targetpolynucleotide purification procedure. In this way, the targetpolynucleotide is substantially purified prior to a subsequent nucleicacid amplification step. Numerous target capture methods are known andsuitable for use in conjunction with the methods described herein.

A “capture oligonucleotide”, “capture oligo”, or “capture probe” refersto a nucleic acid oligomer that specifically hybridizes to a targetsequence in a target nucleic acid by standard base pairing and joins toa binding partner on an immobilized probe to capture the target nucleicacid to a support. One example of a capture oligomer includes twobinding regions: a sequence-binding region (i.e., target-specificportion) and an immobilized probe-binding region, usually on the sameoligomer, although the two regions may be present on two differentoligomers joined together by one or more linkers.

An “immobilized oligo”, “immobilized probe” or “immobilized nucleicacid” refers to a nucleic acid binding partner that joins a captureoligomer to a support, directly or indirectly. An immobilized probejoined to a support facilitates separation of a capture probe boundtarget from unbound material in a sample. Any support may be used, e.g.,matrices or particles free in solution, which may be made of any of avariety of materials, e.g., nylon, nitrocellulose, glass, polyacrylate,mixed polymers, polystyrene, silane polypropylene, or metal.Illustrative examples use a support that is magnetically attractableparticles, e.g., monodisperse paramagnetic beads (uniform size.+−.5%) towhich an immobilized probe is joined directly (e.g., via covalentlinkage, chelation, or ionic interaction) or indirectly (e.g., via alinker), where the joining is stable during nucleic acid hybridizationconditions.

For example, one illustrative approach, as described in U.S. PatentApplication Publication No 20060068417, uses at least one capture probeoligonucleotide that contains a target-complementary region and a memberof a specific binding pair that attaches the target nucleic acid to animmobilized probe on a capture support, thus forming a capture hybridthat is separated from other sample components before the target nucleicacid is released from the capture support.

In another illustrative method, Weisburg et al., in U.S. Pat. No.6,110,678, describe a method for capturing a target polynucleotide in asample onto a solid support, such as magnetically attractable particles,with an attached immobilized probe by using a capture probe and twodifferent hybridization conditions, which preferably differ intemperature only. The two hybridization conditions control the order ofhybridization, where the first hybridization conditions allowhybridization of the capture probe to the target polynucleotide, and thesecond hybridization conditions allow hybridization of the capture probeto the immobilized probe. The method may be used to detect the presenceof a target polynucleotide in a sample by detecting the captured targetpolynucleotide or amplified target polynucleotide.

Another illustrative target capture technique (U.S. Pat. No. 4,486,539)involves a hybridization sandwich technique for capturing and fordetecting the presence of a target polynucleotide. The techniqueinvolves the capture of the target polynucleotide by a probe bound to asolid support and hybridization of a detection probe to the capturedtarget polynucleotide. Detection probes not hybridized to the targetpolynucleotide are readily washed away from the solid support. Thus,remaining label is associated with the target polynucleotide initiallypresent in the sample.

Another illustrative target capture technique (U.S. Pat. No. 4,751,177)involves a method that uses a mediator polynucleotide that hybridizes toboth a target polynucleotide and to a polynucleotide fixed on a solidsupport. The mediator polynucleotide joins the target polynucleotide tothe solid support to produce a bound target. A labeled probe can behybridized to the bound target and unbound labeled pro can be washedaway from the solid support.

Yet another illustrative target capture technique is described in U.S.Pat. Nos. 4,894,324 and 5,288,609, which describe a method for detectinga target polynucleotide. The method utilizes two single-strandedpolynucleotide segments complementary to the same or opposite strands ofthe target and results in the formation of a double hybrid with thetarget polynucleotide. In one embodiment, the hybrid is captured onto asupport.

In another illustrative target capture technique, EP Pat. Pub. No. 0 370694, methods and kits for detecting nucleic acids use oligonucleotideprimers labeled with specific binding partners to immobilize primers andprimer extension products. The label specifically complexes with itsreceptor which is bound to a solid support.

The above capture techniques are illustrative only, and not limiting.Indeed, essentially any technique available to the skilled artisan maybe used provided it is effective for purifying a target nucleic acidsequence of interest prior to amplification.

Nucleic Acid Detection

Essentially any labeling and/or detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the present invention to detect Pseudomonas aeruginosaamplicons. Many such systems are known and available to the skilledartisan, illustrative examples of which are briefly discussed below.

Detection systems typically employ a detection oligo of one type oranother in order to facilitate detection of the target nucleic acid ofinterest. A “detection oligo” or “detection probe” refers to a nucleicacid oligo that hybridizes specifically to a target sequence, includingan amplified sequence, under conditions that promote nucleic acidhybridization, for detection of the target nucleic acid. Detection mayeither be direct (i.e., probe hybridized directly to the target) orindirect (i.e., a probe hybridized to an intermediate structure thatlinks the probe to the target). A probe's target sequence generallyrefers to the specific sequence within a larger sequence which the probehybridizes specifically. A detection probe may include target-specificsequences and other sequences or structures that contribute to theprobe's three-dimensional structure, depending on whether the targetsequence is present (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728,6,835,542, and 6,849,412).

Any of a number of well known labeling systems may be used to facilitatedetection. A “label” refers to a moiety or compound joined directly orindirectly to a probe that is detected or leads to a detectable signal.Direct joining may use covalent bonds or non-covalent interactions(e.g., hydrogen bonding, hydrophobic or ionic interactions, and chelateor coordination complex formation) whereas indirect joining may use abridging moiety or linker (e.g., via an antibody or additionaloligonucleotide(s), which amplify a detectable signal. Any detectablemoiety may be used, e.g., radionuclide, ligand such as biotin or avidin,enzyme, enzyme substrate, reactive group, chromophore such as a dye orparticle (e.g., latex or metal bead) that imparts a detectable color,luminescent compound (e.g. bioluminescent, phosphorescent orchemiluminescent compound), and fluorescent compound. Preferredembodiments include a “homogeneous detectable label” that is detectablein a homogeneous system in which bound labeled probe in a mixtureexhibits a detectable change compared to unbound labeled probe, whichallows the label to be detected without physically removing hybridizedfrom unhybridized labeled probe (e.g., U.S. Pat. Nos. 6,004,745,5,656,207 and 5,658,737). Preferred homogeneous detectable labelsinclude chemiluminescent compounds, more preferably acridinium ester(“AE”) compounds, such as standard AE or AE derivatives which are wellknown (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,948,899). Methods ofsynthesizing labels, attaching labels to nucleic acid, and detectingsignals from labels are well known (e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) at Chapter. 10, and U.S. Pat.Nos. 6,414,152, 5,185,439, 5,658,737, 5,656,207, 5,547,842, 5,639,604,4,581,333, and 5,731,148). Preferred methods of linking an AE compoundto a nucleic acid are known (e.g., U.S. Pat. No. 5,585,481 and U.S. Pat.No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example8). Preferred AE labeling positions are a probe's central region andnear a region of A/T base pairs, at a probe's 3′ or 5′ terminus, or ator near a mismatch site with a known sequence that is the probe shouldnot detect compared to the desired target sequence.

In a preferred embodiment, oligos exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “molecular torches” are designed to includedistinct regions of self-complementarity (coined “the target bindingdomain” and “the target closing domain”) which are connected by ajoining region and which hybridize to one another under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the two complementary regions of the molecular torch, which may be fullyor partially complementary, melt, leaving the target binding domainavailable for hybridization to a target sequence when the predeterminedhybridization assay conditions are restored. Molecular torches aredesigned so that the target binding domain favors hybridization to thetarget sequence over the target closing domain. The target bindingdomain and the target closing domain of a molecular torch includeinteracting labels (e.g., a fluorescent/quencher pair) positioned sothat a different signal is produced when the molecular torch isself-hybridized as opposed to when the molecular torch is hybridized toa target nucleic acid, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized probe having aviable label associated therewith. Molecular torches are fully describedin U.S. Pat. No. 6,361,945, the disclosure of which is herebyincorporated by reference herein.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the invention is a structure commonlyreferred to as a “molecular beacon.” Molecular beacons comprise nucleicacid molecules having a target complementary sequence, an affinity pair(or nucleic acid arms) that holds the probe in a closed conformation inthe absence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe molecular beacon target complementary sequence to the target nucleicacid separates the members of the affinity pair, thereby shifting theprobe to an open conformation. The shift to the open conformation isdetectable due to reduced interaction of the label pair, which may be,for example, a fluorophore and a quencher (e.g., DABCYL and EDANS).Molecular beacons are fully described in U.S. Pat. No. 5,925,517, thedisclosure of which is hereby incorporated by reference herein.Molecular beacons useful for detecting specific nucleic acid sequencesmay be created by appending to either end of one of the probe sequencesdisclosed herein, a first nucleic acid arm comprising a fluorophore anda second nucleic acid arm comprising a quencher moiety. In thisconfiguration, the Pseudomonas aeruginosa-specific probe sequencesdisclosed herein serves as the target-complementary “loop” portion ofthe resulting molecular beacon.

Molecular beacons are preferably labeled with an interactive pair ofdetectable labels. Preferred detectable labels interact with each otherby FRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule or system of molecules by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close such that energy emitted byone label can be received or absorbed by the second label, whether by aFRET or non-FRET mechanism, the two labels are said to be in an “energytransfer relationship.” This is the case, for example, when a molecularbeacon is maintained in the closed state by formation of a stem duplexand fluorescent emission from a fluorophore attached to one arm of themolecular beacon is quenched by a quencher moiety on the other arm.

Illustrative label moieties for the molecular beacons include afluorophore and a second moiety having fluorescence quenching properties(i.e., a “quencher”). In this embodiment, the characteristic signal islikely fluorescence of a particular wavelength, but alternatively couldbe a visible light signal. When fluorescence is involved, changes inemission are preferably due to FRET, or to radiative energy transfer ornon-FRET modes. When a molecular beacon having a pair of interactivelabels in the closed state is stimulated by an appropriate frequency oflight, a fluorescent signal is generated at a first level, which may bevery low. When this same molecular beacon is in the open state and isstimulated by an appropriate frequency of light, the fluorophore and thequencher moieties are sufficiently separated from each other such thatenergy transfer between them is substantially precluded. Under thatcondition, the quencher moiety is unable to quench the fluorescence fromthe fluorophore moiety. If the fluorophore is stimulated by light energyof an appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used in connectionwith the invention, making no attempt to distinguish FRET from non-FRETpairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/DABCYL, coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPYFL, fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL,eosine/DABCYL, erythrosine/DABCYL, tetramethylrhodamine/DABCYL, TexasRed/DABCYL, CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2, and fluorescein/QSY7dye. Those having an ordinary level of skill in the art will understandthat when donor and acceptor dyes are different, energy transfer can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor speciesare the same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BlackHole Quencher moieties, which are available from Biosearch Technologies,Inc. (Novato, Calif.).

Synthetic techniques and methods of attaching labels to nucleic acidsand detecting labels are well known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. Nos.5,185,439 and 6,004,745; Kourilsky et al., U.S. Pat. No. 4,581,333; and,Becker et al., U.S. Pat. No. 5,731,148).

Preferred Pseudomonas Aeruginosa Oligos and Oligo Sets

As described herein, preferred sites for amplifying and detectingPseudomonas aeruginosa nucleic acids according to the present inventionhave been found to reside in the 800 region of Pseudomonas aeruginosa23s rRNA. Moreover, particularly preferred oligonucleotides andoligonucleotide sets within this region have been identified foramplifying Pseudomonas aeruginosa 23s with improved sensitivity,selectivity and specificity. It will be understood that theoligonucleotides of the invention are capable of hybridizing to aPseudomonas aeruginosa target sequence with high specificity and, as aresult, are capable of participating in a nucleic acid amplificationreaction that can be used to detect the presence and/or levels ofPseudomonas aeruginosa in a sample and distinguish it from the presenceof other pseudomonads.

For example, in one embodiment, the amplification oligonucleotides ofthe invention comprise a first oligonucleotide and a secondoligonucleotide, wherein the first and second oligonucleotides targetthe 800 region of the Pseudomonas aeruginosa 23s rRNA with a high degreeof specificity. Of course, it will be understood, when discussing theamplification oligonucleotides of the invention, that the first andsecond oligonucleotides used in an amplification reaction havespecificity for opposite strands of the target nucleic acid sequence tobe amplified.

In a particular embodiment, the amplification oligonucleotides of theinvention comprise a first oligonucleotide and a second oligonucleotide,wherein the first oligonucleotide targets the complement of a sequencein a region of Pseudomonas aeruginosa 23s rRNA corresponding to basesfrom about 725-825 of E. coli 23s rRNA, and the second oligonucleotidetargets a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 845-950 of E. coli 23s rRNA.

The amplification oligonucleotides of the invention are particularlyeffective for amplifying a target nucleic acid sequence of Pseudomonasaeruginosa in a transcription-based amplification reaction, preferably atranscription-mediated amplification (TMA) reaction.

Certain amplification oligonucleotides of the invention are used in atranscription-mediated amplification reaction and comprise a T7 provideroligonucleotide and a non-T7 primer oligonucleotide, wherein the T7provider targets the complement of a sequence in a region of Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 725-825 of E. coli23s rRNA, and the non-T7 primer targets a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about845-950 of E. coli 23s rRNA.

Certain more specific amplification oligonucleotides of the inventioncomprise a T7 provider oligonucleotide and a non-T7 primeroligonucleotide, wherein the T7 provider targets the complement of asequence in a region of Pseudomonas aeruginosa 23s rRNA corresponding tobases from about 725-775 of E. coli 23s rRNA, and the non-T7 primertargets a sequence in a region of the Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 900-950 of E. coli 23s rRNA.

Other specific amplification oligonucleotides of the invention comprisea T7 provider oligonucleotide and a non-T7 primer oligonucleotide,wherein the T7 provider targets the complement of a sequence in a regionof Pseudomonas aeruginosa 23s rRNA corresponding to bases from about739-766 of E. coli 23s rRNA, and the non-T7 primer targets Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 918-943 of E. coli23s rRNA.

In a specific and preferred embodiment, the amplificationoligonucleotides of the invention comprise a T7 provider oligonucleotideand a non-T7 primer oligonucleotide, wherein the T7 provider is selectedfrom SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:11 or SEQ ID NO:14, and thenon-T7 primer is selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:19or SEQ ID NO:15.

In another preferred embodiment, the amplification oligonucleotides ofthe invention comprise a T7 provider oligonucleotide and a non-T7 primeroligonucleotide, wherein the T7 provider is SEQ ID NO:2 and the non-T7primer is SEQ ID NO:24.

It will be understood that in addition to the particular T7 provideroligonucleotides and non-T7 primer oligonucleotides used in theamplification reaction, additional oligonucleotides will also generallybe employed in conjunction with the amplification reaction. For example,in certain embodiments, the amplification reactions will also employ theuse of one or more of a detection oligonucleotide (e.g., a torcholigonucleotide), a blocker oligonucleotide and/or an extendoligonucleotide.

Table 1 below presents specific examples of T7 provideroligonucleotides, non-T7 primer oligonucleotides, and other ancillaryoligonucleotides (e.g., blocker oligonucleotides, extendoligonucleotides and torch oligonucleotides) that have been identifiedby the present invention as having particularly preferred features.

TABLE 1 Examples of Preferred Oligos Oligo Description Oligo IDSequence 5′-3′ T7 provider SEQ ID NO: 2 AATTTAATACGACTCACTATAGGGAGACGTTGAAAAGGTAGGGGATGACTTGTGG X SEQ ID NO: 1 AATTTAATACGACTCACTATAGGGAGAGAACCCACTCCCGTTGAAAAGGTAG G X SEQ ID NO: 11 AATTTAATACGACTCACTATAGGGAGACTCGGAGATAGCTGGTTCTCCTCGAAAGC X SEQ ID NO: 14AATTTAATACGACTCACTATAGGGAGA GCTGGTTCTCCTCGAAAGC X Non-T7 primerSEQ ID NO: 22 ccauGCTCGGCACTTCTGGGTATTCG SEQ ID NO: 24GTGTGTCTCCCATGCTCGGCACTTCTG SEQ ID NO: 19 cgguTTGGTAAGTCGGGATGACCCSEQ ID NO: 15 ccuaGCCGAAACAGTTGCTCTACCC Torch SEQ ID NO: 51cccagagugauacaugcuggg SEQ ID NO: 54 cccagagugauaccuggg SEQ ID NO: 50gccucagagugauacaugaggc SEQ ID NO: 56 cccagagugauacaugagcuggg BlockerSEQ ID NO: 29 caacgggaguggguucggu X SEQ ID NO: 26 gguucgguccuccagucag XSEQ ID NO: 40 ccgagcuugauuagccuuucacuccg X SEQ ID NO: 42ccagcuaucuccgagcuugauuagc X Extend Oligo SEQ ID NO: 43GGATGACTTGTGGATCGGAGTGAAAGG X SEQ ID NO: 44ATCGGAGTGAAAGGCTAATCAAGCTCG X Lower case 2′-O--methyl RNA X is ablocking moiety (e.g., reverse (3′-5′) C blocked)

In addition, Table 2 below identifies a particularly preferredoligonucleotide set for use in the compositions, kits and methods of thepresent invention, which comprises the T7 provider oligonucleotide SEQID NO:2 and the non-T7 primer oligonucleotide, SEQ ID NO:24, andoptionally further comprises the blocker oligonucleotide SEQ ID NO:29,the torch oligonucleotide SEQ ID NO:54, the extend oligonucleotide SEQID NO:44, the target capture oligonucleotide SEQ ID NO:69 and,optionally, the target capture helper oligonucleotide SEQ ID NO:73.

TABLE 2 Example of Preferred Oligo Set Oligo Description Oligo IDSequence 5′-3′ Torch SEQ ID NO: 54 cccagagugauaccuggg T7 providerSEQ ID NO: 2  AATTTAATACGACTCACTATAGGGAGA CGTTGAAAAGGTAGGGGATGACTTGTGG XBlocker SEQ ID NO: 29 caacgggaguggguucggu X Non-T7 Primer SEQ ID NO: 24GTGTGTCTCCCATGCTCGGCACTTCTG Extend Oligo SEQ ID NO: 44ATCGGAGTGAAAGGCTAATCAAGCTCG X Target Capture SEQ ID NO: 68gcuccucuaccgcgucacuuacg-dT₃dA₃₀ Oligo Target Capture SEQ ID NO: 73cucaacucaccuucacaggcuuacagaacX Oligo Helper Lower case 2′-O--methyl RNAX is a blocking moiety (e.g., reverse (3′-5′) C blocked)

While specifically preferred amplification oligonucleotides derived fromthe 800 region have been identified according to the invention, whichresult in superior assay performance, it will be recognized that otheroligonucleotides derived from the 800 region and having insubstantialmodifications from those specifically described herein may also be used,provided the same or similar performance objectives are achieved. Forexample, oligonucleotides derived from the 800 region and useful in theamplification reactions according to the invention can have differentlengths from those identified herein, provided it does not substantiallyaffect amplification and/or detection procedures. These and otherroutine and insubstantial modifications to the preferredoligonucleotides of the invention can carried out using conventionaltechniques, and to the extent such modifications maintain one or moreadvantages provided herein they are considered within the spirit andscope of the present invention.

Preferred Compositions and Kits for Detecting Pseudomonas Aeruginosa

The present invention also embraces compositions, reaction mixtures andkits for performing polynucleotide amplification reactions for detectingthe 800 region of the 23s rRNA of Pseudomonas aeruginosa. Exemplary kitsinclude first and second amplification oligonucleotides that arecomplementary to opposite strands of the 800 region of the 23s rRNA ofPseudomonas aeruginosa. Certain preferred kits will containoligonucleotides described herein for use in a transcription-associatedamplification reaction, preferably a TMA reaction. In one preferredembodiment, a kit of the invention will comprise a T7 provideroligonucleotide as described herein, a non-T7 primer oligonucleotide asdescribed herein, and optionally will further comprise one or more otherancillary oligonucleotides to facilitate amplification and/or detection,including one or more of a detection oligonucleotide, captureoligonucleotide, blocker oligonucleotide, and/or extend oligonucleotide,as described herein.

In certain embodiments, the present invention is drawn to compositions,reaction mixtures and kits comprising a first oligonucleotide and asecond oligonucleotide, wherein the first and second oligonucleotidestarget the 800 region of the Pseudomonas aeruginosa 23s rRNA with highspecificity. Of course, it will be understood, when discussing theamplification oligonucleotides of the invention that target certainresidues within this 800 region, that the first and secondoligonucleotides used in an amplification reaction are complementary toopposite strands of the target nucleic acid sequence to be amplified.

In a particular embodiment, the present invention is drawn tocompositions, reaction mixtures and kits, for use in an amplificationreaction, comprising a first oligonucleotide and a secondoligonucleotide, wherein the first oligonucleotide targets thecomplement of a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 725-825 of E. coli 23s rRNA, and thesecond oligonucleotide targets a sequence in a region of Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 845-950 of E. coli23s rRNA.

In another embodiment, the present invention is drawn to compositions,reaction mixtures and kits, for use in a transcription mediatedamplification reaction, comprising a T7 provider oligonucleotide and anon-T7 primer oligonucleotide, wherein the T7 provider targets thecomplement of a sequence in a region of Pseudomonas aeruginosa 23s rRNAcorresponding to bases from about 725-825 of E. coli 23s rRNA, and thenon-T7 primer targets a sequence in a region of the Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 845-950 of E. coli23s rRNA.

In a more particular embodiment, the present invention is drawn tocompositions, reaction mixtures and kits, for use in a transcriptionmediated amplification reaction, comprising a T7 provideroligonucleotide and a non-T7 primer oligonucleotide, wherein the T7provider targets the complement of a sequence in a region of Pseudomonasaeruginosa 23s rRNA corresponding to bases from about 725-775 of E. coli23s rRNA, and the non-T7 primer targets a sequence in a region ofPseudomonas aeruginosa 23s rRNA corresponding to bases from about900-950 of E. coli 23s rRNA.

In a more specific embodiment, the present invention is drawn tocompositions, reaction mixtures and kits, for use in a transcriptionmediated amplification reaction, comprising a T7 provideroligonucleotide and a non-T7 primer oligonucleotide, wherein the T7provider targets the complement of a sequence in a region of thePseudomonas aeruginosa 23s rRNA corresponding to bases from about739-766 of E. coli 23s rRNA, and the non-T7 primer targets a sequence ina region of the Pseudomonas aeruginosa 23s rRNA corresponding to basesfrom about 918-943of E. coli 23s rRNA.

In one preferred embodiment, the present invention is drawn tocompositions, reaction mixtures and kits, for use in a transcriptionmediated amplification reaction, comprising at least a T7 provideroligonucleotide and a non-T7 primer oligonucleotide, wherein the T7provider is selected from SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:11 or SEQID NO:14, and the non-T7 primer is selected from SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:19 or SEQ ID NO:15.

In another preferred embodiment, the present invention is drawn tocompositions, reaction mixtures and kits, for use in a transcriptionmediated amplification reaction, comprising at least a T7 provideroligonucleotide and a non-T7 primer oligonucleotide, wherein the T7provider is SEQ ID NO:2 and the non-T7 primer is SEQ ID NO:24.

It will be understood that in addition to the particular T7 provideroligonucleotides and non-T7 primer oligonucleotides used in theamplification reaction, additional ancillary oligonucleotides will alsogenerally be employed in conjunction with the amplification reaction.For example, in certain embodiments, the compositions, reaction mixturesand kits of the invention will also further comprise one or more of atarget capture oligonucleotide, torch oligonucleotide, blockeroligonucleotide and/or extend oligonucleotide.

For example, in one embodiment, the compositions, reaction mixturesand/or kits of the invention, in addition to the T7 provideroligonucleotide and non-T7 primer oligonucleotide, will further comprisea torch oligonucleotide. In a particular embodiment, the torcholigonucleotide is selected from SEQ ID NO:51, SEQ ID NO:54, SEQ IDNO:50 or SEQ ID NO:56. In one preferred embodiment, the torcholigonucleotide is SEQ ID NO:54.

In another embodiment, the compositions, reaction mixtures and/or kitsof the invention, in addition to the T7 provider oligonucleotide andnon-T7 primer oligonucleotide, will further comprise an extendoligonucleotide. In a particular embodiment, the extend oligonucleotideis selected from SEQ ID NO:43 or SEQ ID NO: 44.

In another embodiment, the compositions, reaction mixtures and/or kitsof the invention, in addition to the T7 provider oligonucleotide andnon-T7 primer oligonucleotide, will further comprise a blockeroligonucleotide. In a particular embodiment, the blocker oligonucleotideis selected from SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:40 or SEQ IDNO:42.

In another embodiment, the compositions, reaction mixtures and/or kitsof the invention, in addition to the T7 provider oligonucleotide andnon-T7 primer oligonucleotide, will further comprise a target captureoligonucleotide and, optionally, a target capture helperoligonucleotide. In a particular embodiment, the target captureoligonucleotide SEQ ID NO:69 and the target capture helperoligonucleotide is SEQ ID NO:73.

In a particularly preferred embodiment, the compositions, reactionmixtures and/or kits of the invention comprise the T7 provideroligonucleotide, SEQ ID NO:2 and the non-T7 primer oligonucleotide, SEQID NO:24; and optionally further comprises the blocker oligonucleotideSEQ ID NO:29, the torch oligonucleotide SEQ ID NO:54, the extendoligonucleotide SEQ ID NO:44, the target capture oligonucleotide SEQ IDNO:69, and optionally, the target capture helper oligonucleotide SEQ IDNO:73.

The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting Examples.

EXAMPLES

Examples are provided below illustrating certain aspects and embodimentsof the invention. The examples below are believed to accurately reflectthe details of experiments actually performed, however, it is possiblethat some minor discrepancies may exist between the work actuallyperformed and the experimental details set forth below which do notaffect the conclusions of these experiments or the ability of skilledartisans to practice them. Skilled artisans will appreciate that theseexamples are not intended to limit the invention to the specificembodiments described therein. Additionally, those skilled in the art,using the techniques, materials and methods described herein, couldeasily devise and optimize alternative amplification systems forcarrying out these and related methods while still being within thespirit and scope of the present invention.

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. See e.g., Carruthers, et al., 154 Methods inEnzymology, 287 (1987), the contents of which are hereby incorporated byreference herein. Unless otherwise stated herein, modified nucleotideswere 2′-O-methyl ribonucleotides, which were used in the synthesis astheir phosphoramidite analogs. For blocked oligonucleotides used insingle-primer amplification (Becker et al., US2006/0046265, herebyincorporated by reference herein), the 3′-terminal blocking moietyconsisted of a “reversed C” 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Cat. No. 20-0102-01).Molecular torches (see Becker et al., U.S. Pat. No. 6,849,412, herebyincorporated by reference herein) were prepared using a C9non-nucleotide linker joining region, 5′-fluorescein (“F”) fluorophoreand 3′-dabsyl (“D”) quencher moieties attached to the oligonucleotide bystandard methods.

As set forth in the examples below, analyses of a wide variety ofamplification reagents and conditions has led to the development of ahighly sensitive and selective amplification process for thespecies-specific detection of Pseudomonas aeruginosa.

Example 1 Description of Illustrative Assay Reagents and Protocols

The following example describes typical assay reagents, protocols,conditions and the like used in the TMA experiments described herein.Unless specified to the contrary, reagent preparation, equipmentpreparation and assay protocols were performed essentially as set forthbelow.

A. Reagents and Samples

1. Amplification Reagent. The “Amplification Reagent” or “Amp Reagent”comprised approximate concentrations of the following components: 0.5 mMdATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 10 mM ATP, 2 mM CTP, 2 mMGTP, 12.7 mM UTP, 30 mM MgCl₂, and 33 mM KCl in 50 mM HEPES buffer at pH7.7. Primers and other oligonucleotides were added to the Amp Reagent.

2. Enzyme Reagent. The “Enzyme Reagent” comprised approximateconcentrations of the following components: 1180 RTU/μL Moloney murineleukemia virus (“MMLV”) reverse transcriptase (“RT”) and 260 PU/μL T7RNA polymerase in 75 mM HEPES buffer containing 120 mM KCl, 10% TRITON®X-100, 160 mM N-acetyl-L-cysteine, and 1 mM EDTA at pH 7.0, where oneRTU of RT activity incorporates 1 nmol of dT into a substrate in 20minutes at 37° C. and one PU of T7 RNA polymerase activity produces 5fmol of RNA transcript in 20 minutes at 37° C.

3. Wash Solution. The “Wash Solution” comprised 0.1% (w/v) sodiumdodecyl sulfate, 150 mM NaCl and 1 mM EDTA in 10 mM HEPES buffer at pHto 7.5.

4. Target Capture Reagent. The “Target Capture Reagent” (TCR) comprisedapproximate concentrations of the following components: 60 pmol/mL eachof one or more capture probes having a dT₃dA₃₀ tail and an optionalcapture helper probe, 250 to 300 ug/mL paramagnetic oligo-(dT)₁₄microparticles (Seradyn), 250 mM HEPES, 100 mM EDTA and 1.88 M LiCl atpH 6.5.

5. Lysis Reagent. The “Lysis Buffer” comprised 1% lithium lauryl sulfatein a buffer containing 100 mM tris, 2.5 mM succinic acid, 10 mM EDTA and500 mM LiCl at pH 6.5.

6. Target rRNA Samples. rRNA samples were stored in water, 0.1% LiLS orLysis Reagent prior to use in the experiments described herein.

B. Target Capture

A typical target capture procedure to purify and prepare nucleic acidsamples for subsequent amplification is performed essentially asdescribed below.

1. Combine 100 uL of test sample, 50 μL of the TCR containing targetcapture oligonucleotides and 1 mL Lysis Reagent. Incubate the mixture at60° C. for 15 minutes

2. Capture and wash the TCR magnetic particles from the treated reactionmixture using the Wash Solution and a suitable magnetic particle washingand separation device (e.g., a magnetic separation rack, a GEN-PROBE®Target Capture System, Gen-Probe Cat. No. 5207, or a KingFisher®magnetic particle processor system available from Thermo Labsystems).

3. After washing, the magnetic particles are resuspended in 100 μL ofthe Amplification Reagent.

C. Amplification and Detection of Target

The real-time TMA amplification reactions were performed essentially asfollows. 30 μL of sample, amplification and detection oligonucleotidesin the Amp Reagent or 30 μL of the resuspended particles in the AmpReagent from the target capture procedure was incubated at 60° C. for 10minutes. The temperature was then reduced and the reaction mixture wasequilibrated to 42° C. 10 μL of Enzyme Reagent was added. The reactionmixture was mixed and incubated at 42° C. in a real-time detectionsystem (e.g., Opticon™ or Chromo4™ detection systems available fromBio-Rad Laboratories, or a FluoDia® T70 instrument).

Example 2 Design and Initial Testing of Pseudomonas Aeruginosa (Pae)Oligo Sets

Amplication and detection oligonucleotides targeting two regions ofPseudomonas aeruginosa nucleic acid corresponding to from about 700 to1000 (“800 region”) and from about 1400 to 1600 (“1500 region”)nucleotide base positions of E. coli 23s rRNA (Accession No. V00331)were designed and synthesized for evaluation.

TABLE 3 800 Region oligos SEQ ID Use NO: Sequence (5′-3′) T7 Provider  1AATTTAATACGACTCACTATAGGGAGA GAACCCACTCCCGTTGAAAAGGTAGG-X T7 Provider  2AATTTAATACGACTCACTATAGGGAGA CGTTGAAAAGGTAGGGGATGACTTGTGG-X T7 Provider 3 AATTTAATACGACTCACTATAGGGAGA GGATGACTTGTGGATCGGAGTGAAAGGCTAATC-XT7 Provider  4 AATTTAATACGACTCACTATAGGGAGAGTGGATCGGAGTGAAAGGCTAATCAAGCTC-X T7 Provider  5AATTTAATACGACTCACTATAGGGAGA TCGGAGTGAAAGGCTAATCAAGCTCGGAGATAG-XT7 Provider  6 AATTTAATACGACTCACTATAGGGAGATCGGAGTGAAAGGCTAATCAAGCTCGGAG-X T7 Provider  7AATTTAATACGACTCACTATAGGGAGA GAGTGAAAGGCTAATCAAGCTCGGAGATAGCTG-XT7 Provider  8 AATTTAATACGACTCACTATAGGGAGAGGCTAATCAAGCTCGGAGATAGCTGGTTC-X T7 Provider   9AATTTAATACGACTCACTATAGGGAGA GGCTAATCAAGCTCGGAGATAGCTGGTTCTCC-XT7 Provider 10 AATTTAATACGACTCACTATAGGGAGAATCAAGCTCGGAGATAGCTGGTTCTCCTCGAA-X T7 Provider 11AATTTAATACGACTCACTATAGGGAGA CTCGGAGATAGCTGGTTCTCCTCGAAAGC-X T7 Provider12 AATTTAATACGACTCACTATAGGGAGA GGAGATAGCTGGTTCTCCTCGAAAGCTATTTA-XT7 Provider 13 AATTTAATACGACTCACTATAGGGAGAAGCTGGTTCTCCTCGAAAGCTATTTAGGTAG-X T7 Provider 14AATTTAATACGACTCACTATAGGGAGA GCTGGTTCTCCTCGAAAGC-X Primer 15ccuaGCCGAAACAGTTGCTCTACCC Primer 16 gucgGGATGACCCCCTAGCCGAAACAGTTGPrimer 17 gucgGGATGACCCCCTAGCCGAAACAG Primer 18gguaAGTCGGGATGACCCCCTAGCCGAAA Primer 19 cgguTTGGTAAGTCGGGATGACCC Primer20 guauTCGGAGTTTGCATCGGTTTGGTA Primer 21 CTTCTGGGTATTCGGAGTTTGCATCGGTTTGPrimer 22 ccauGCTCGGCACTTCTGGGTATTCG Primer 23augcTCGGCACTTCTGGGTATTCGGAG Primer 24 GTGTGTCTCCCATGCTCGGCACTTCTGBlocker 25 cgguccuccagucaguguuac-X Blocker 26 gguucgguccuccagucag-XBlocker 27 gaguggguucgguccuccag-X Blocker 28 gggaguggguucgguccucc-XBlocker 29 caacgggaguggguucggu-X Blocker 30 cuuuucaacgggaguggguuc-XBlocker 31 cauccccuaccuuuucaacgggagu-X Blocker 32 cuaccuuuucaacgggagug-XBlocker 33 uccacaagucauccccuaccuuuuc-X Blocker 34uccgauccacaagucauccccuacc-X Blocker 35 cuccgauccacaagucauccccua-XBlocker 36 cacuccgauccacaagucauccccu-X Blocker 37ucacuccgauccacaagucaucc-X Blocker 38 uagccuuucacuccgauccacaagu-X Blocker39 cuugauuagccuuucacuccgaucc-X Blocker 40 ccgagcuugauuagccuuucacuccg-XBlocker 41 ucuccgagcuugauuagccuuucac-X Blocker 42ccagcuaucuccgagcuugauuagc-X Extend oligo 43GGATGACTTGTGGATCGGAGTGAAAGG-X Extend oligo 44ATCGGAGTGAAAGGCTAATCAAGCTCG-X Extend oligo 45CTGGTTCTCCTCGAAAGCTATTTAG-X Torch 46 ccgagugauacaugaggcgcucgg Torch 47cccagagugauacaugaggcgcuggg Torch 48 cccagagugauacaugaggcgcuggg Torch 49ccagagugauacaugaggcucugg Torch 50 gccucagagugauacaugaggc Torch 51cccagagugauacaugcuggg Torch 52 cccagagugauacaucuggg Torch 53cccagagugauacacuggg Torch 54 cccagagugauaccuggg Torch 55cccagagugauacuggg Torch 56 cccagagugauacaugagcuggg Torch 57cuaccccccagagagauggguag

TABLE 4 1500 Region Oligos SEQ ID Use NO: Sequence (5′-3′) Blocker 58cacccacggccaagcgg-X Blocker 59 ggauuuaccuaagauuucag-XAATTTAATACGACTCACTATAGGGAGA T7 Provider 60 GGGTGGCCAAGTTTAAGGTGGTAGGC-XAATTTAATACGACTCACTATAGGGAGA T7 Provider 61 GGTAAATCCGGGGTTTCAAGGCCG-XTorch 62 cgacgacucgucaucacgucg Primer 63 ggaaGCATGGCATCAAGCAC Primer 64 ccugGTTACCTGAAGCTTAGAAGCTTTTCTTGG

The 23S 1500 region designs were tested with results presented below inCycle Time which is proportional to Ttime. Four combinations of oligoswere tested with oligo set number two being the best candidate based onthe curve shape (not shown) and earliest Cycle Time at 1E+06 copies ofPae rRNA.

TABLE 5 Summary of Oligo Screening for Pae 23S 1500 Region Cycle CycleTime @ SEQ Time @ 1E+06 ID 0 copies copies Set Description NO: RNA RNAComments 1) T7 provider 60 62.5 25.2 Low Blocker 58 None 52.1amplification @ Non-T7 primer 63 66.5 54.9 both copy levels Torch 6246.6 53.7 2) T7 provider 60 47.7 32.5 Best set even with Blocker 58 49.420.9 contamination of Non-T7 primer 64 52.2 32.8 reagents Torch 62 52.433.9 3) T7 provider 61 None 55.8 Low Blocker 59 None 57.1 amplification@ Non-T7 primer 63 None 53.1 both copy levels Torch 62 25.6 56.7 4) T7provider 61 67.7 46.0 Second best set Blocker 59 57.8 39.8 even withNon-T7 primer 64 26.6 40.5 contamination of Torch 62 7.4 41.9 reagents

Several optimization experiments with the 1500 region oligo set two wereperformed varying the concentrations of the oligos (results not shown).However, simultaneous testing with the long amplicon oligo set(s) in the800 region showed a reduction in the level of background with thecontaminated reagents. Accordingly, the 800 region of the 23S rRNA wasselected as the preferred region for further optimization based upon thefinding that T7 providers and non-T7 primers for this region displayedthe highest signals and lowest background in a real-time single primerTMA assay, relative to the large number of other oligo sets tested.Screening of oligos in a real-time TMA assay was performed, anddifferent torches and blockers were also analyzed. The criteria forselecting the best oligo sets included having the lowest background andthe highest signal at 10e3 copies of Pseudomonas aeruginosa rRNA.

Three initial preferred oligo sets were selected from this analysis,including blockers and torches, and these sets are referred to as thestandard short amplicon sets S1 and F11, and the long amplicon set LA2(Table 6 below). Although each of the three sets performed well, the LA2oligo set demonstrated no background and the best sensitivity. Inaddition, the LA2 oligo set was unusual as the amplicon produced, atabout 196 bases in length, is longer than typical shorter amplicons(86-110 nucleotide bases) of F11 and S1.

TABLE 6 Summary Table of Initial 800 Region Oligos Sets Tested Oligo SetDescription Oligo ID S1 T7 provider SEQ ID NO: 11 Blocker SEQ ID NO: 40Torch SEQ ID NO: 56 Non-T7 primer SEQ ID NO: 19 F11 T7 provider SEQ IDNO: 14 Blocker SEQ ID NO: 42 Torch SEQ ID NO: 51 Non-T7 primer SEQ IDNO: 15 LA2 T7 provider SEQ ID NO: 2 Blocker SEQ ID NO: 29 Torch SEQ IDNO: 50 Non-T7 primer SEQ ID NO: 22

Example 3 Further Identification of Pseudomonas Aeruginosa Oligo Sets

To further reduce background signals and improve specificity andsensitivity, a number of additional oligo sets were designed and tested.“Extend” oligos were also introduced in an effort to improvesensitivity. These experiments were performed without target capture.

Prior to the addition of the extend oligos and redesigns, one oligo setbeing tested (designated as the “standard” system) comprised acombination of the amplification oligos from the “LA2” oligo set and thetorch from the “F11” oligo set (Table 7 below). This oligo set, however,exhibited specificity problems when challenged with Pseudomonas putida,had slightly elevated background signals, and had slow emergence timesfor Pseudomonas aeruginosa at 1×10³ copies. Redesign and screeningyielded three promising new oligo sets (Sets 1-3 below), which addressedthese problems. All three oligo sets included the torch SEQ ID NO:54,which improved specificity and reduced background signals; extend oligosfor improving assay sensitivity; and redesigned amplification oligos.

In addition, these new oligo sets were compared with another oligo set(Set 4 below) and the “standard LA” oligo set. All of the new oligo setsincluded torch SEQ ID NO:54, and they outperformed the “standard” oligoset.

Table 7 below summarizes the preferred oligo sets at this stage of theanalysis. Table 7 below shows the sequences of preferred the T7providers, non-T7 primers, extend oligos and blocker oligos.

TABLE 7 Summary of Certain Preferred Long Amplicon Oligo Sets Oligo SetDescription Oligo “Standard LA” Set Torch SEQ ID NO: 51 (LA2 + F11torch) T7 provider SEQ ID NO: 2 Blocker SEQ ID NO: 29 Non-T7 primer SEQID NO: 22 Set 1 Torch SEQ ID NO: 54 T7 provider SEQ ID NO: 2 Blocker SEQID NO: 25 Extend Oligo SEQ ID NO: 44 Non-T7 primer SEQ ID NO: 24 Set 2Torch SEQ ID NO: 54 T7 provider SEQ ID NO: 2 Blocker SEQ ID NO: 29Extend Oligo SEQ ID NO: 44 Non-T7 primer SEQ ID NO: 22 Set 3 Torch SEQID NO: 54 T7 provider SEQ ID NO: 1 Blocker SEQ ID NO: 26 Extend OligoSEQ ID NO: 43 Non-T7 primer SEQ ID NO: 24 Set 4 Torch SEQ ID NO: 54 T7provider SEQ ID NO: 1 Blocker SEQ ID NO: 26 Extend Oligo SEQ ID NO: 43Non-T7 primer SEQ ID NO: 22

TABLE 8 Sequences for Preferred Oligos Description Oligo Sequence 5′-3′Torch SEQ ID NO: 51 cccagagugauacaugcuggg SEQ ID NO: 54cccagagugauaccuggg T7 provider SEQ ID NO: 2 AATTTAATACGACTCACTATAGGGAGACGTT GAAAAGGTAGGGGATGACTTGTGG-X SEQ ID NO: 1 AATTTAATACGACTCACTATAGGGAGAGAAC CCACTCCCGTTGAAAAGGTAGG X Blocker SEQ ID NO: 29caacgggaguggguucggu X SEQ ID NO: 26 gguucgguccuccagucag X Non-T7 primerSEQ ID NO: 22 ccauGCTCGGCACTTCTGGGTATTCG SEQ ID NO: 24GTGTGTCTCCCATGCTCGGCACTTCTG Extend Oligo SEQ ID NO: 43GGATGACTTGTGGATCGGAGTGAAAGG X SEQ ID NO: 44ATCGGAGTGAAAGGCTAATCAAGCTCG X

Example 4 Further Characterization and Optimization of PseudomonasAeruginosa Oligo Sets

Tables 9-13 below present and summarize representative data relating tothe identification and optimization of certain preferred oligo sets ofthe present invention. The AveRange (RFU) and TTime (min) results fromreal-time TMA reactions are presented. Preferred oligos are determinedfrom this analysis by the curve shape (not shown) and the results forthe AveRange and TTime; the preferred oligo sets have the lowestrelative fluorescence unit (RFU) and the longest TTime at the zero PaerRNA copy level. High RFU values at the zero Pae rRNA copy levelindicate possible contamination within the reagents.

Table 9 presents a summary of results for oligo set S1.

TABLE 9 Summary of results for oligo set S1 Oligo Copies Pae AveRangeTTime Set rRNA (RFU) (min) Comments S1 0 0.341 20.6 AveRange and TTime1E+04 0.393 16.7 @ 0 copies Pae rRNA 1E+05 0.297 14.8 indicates 1E+060.326 12.6 contamination 1E+07 0.417 10.8 1E+08 0.449 9.1

Table 10 compares the S1 oligo set with the F11 oligo set anddemonstrates for F11 the first reduction in RFU at the zero copy level.This finding was built upon for subsequent testing.

TABLE 10 Summary of results for oligo set S1 vs. F11 Oligo Copies PaeAveRange TTime Set rRNA (RFU) (min) Comments S1 0 0.745 18.9Contamination 1E+03 0.757 19.0 F11 pure 0 0.503 33.8 Contaminationreduced 1E+03 0.727 29.2 F11 crude 0 0.426 47.0 Contamination reduced1E+03 1.185 37.9

The oligo set, LA2, as well as combinations of previously screenedoligos, were tested and reductions in RFU values at the 0 copy levelwere also demonstrated (e.g., Table 11). The LA2 oligo set produces amuch longer amplicon than for previous sets tested and the level ofPseudomonas aeruginosa contamination at the zero P. aeruginosa rRNA copylevel was decreased even further (Table 11).

TABLE 11 Summary Table of LA2 & Combinations Copies Ave Oligo Oligo PaeRange TTime Set Description (ID) rRNA (RFU) (min) Comments Control T7provider 0 0.389 14.9 (F11 amp + (SEQ ID NO: 14) S1 torch) Blocker 1E+040.298 13.6 (SEQ ID NO: 42) Torch 1E+06 0.324 10.9 (SEQ ID NO: 56) Primer(SEQ ID NO: 15) Combo2 T7 provider 0 0.183 34.6 (LA2) (SEQ ID NO:2)Blocker 1E+04 0.344 27.0 (SEQ ID NO: 29) Torch 1E+06 0.370 19.9 (SEQ IDNO: 50) Primer (SEQ ID NO: 22) Combo3 T7 provider 0 0.042 29.5 Lower(SEQ ID NO: 2) RFU & Blocker 1E+04 0.312 23.7 later (SEQ ID NO: 29)TTime @ Torch 1E+06 0.308 17.9 0 copies (SEQ ID NO: 56) 1 Primer (SEQ IDNO: 5) Combo4 T7 provider 0 0.121 21.1 (SEQ ID NO: 14) Blocker 1E+040.305 17.9 (SEQ ID NO: 42) Torch 1E+06 0.378 13.9 (SEQ ID NO: 56) Primer(SEQ ID NO: 22) Combo5 T7 provider 0 0.088 28.7 (SEQ ID NO: 2) Blocker1E+04 0.284 22.9 (SEQ ID NO: 29) Torch 1E+06 0.452 17.6 (SEQ ID NO: 50)Primer (SEQ ID NO: 15) Combo6 T7 provider 0 0.476 19.8 (SEQ ID NO: 14)Blocker 1E+04 0.350 17.5 (SEQ ID NO: 42) Torch 1E+06 0.422 13.5 (SEQ IDNO: 50) Primer (SEQ ID NO: 22) Combo7 T7 provider 0 0.567 14.2 (SEQ IDNO: 14) Blocker 1E+04 0.352 12.8 (SEQ ID NO: 42) Torch 9 1E+06 0.45010.1 SEQ ID NO: 50) Primer (SEQ ID NO: 15) Combo8 T7 provider 0 0.01439.1 Lowest (SEQ ID NO: 2) RFU & Blocker 1E+04 0.211 28.4 latest (SEQ IDNO: 29) TTime @ Torch 1E+06 0.302 22.0 0 copies (SEQ ID NO: 56) Primer(SEQ ID NO: 22)In addition, various torches were tested with the LA2 oligo set and theresults are set forth in Table 12.

TABLE 12 Summary Table for various Non-T7 primer and Torch combinationsCopies Ave Oligo Oligo Pae Range TTime Set Description (ID) rRNA (RFU)(min) Comments LA1 T7 provider (SEQ ID NO: 2) 0 0.090 22.2 Blocker (SEQID NO: 29) 1E+02 0.112 20.3 Torch (SEQ ID NO: 50) 1E+03 0.260 21.0Non-T7 primer (SEQ ID NO: 15) 1E+04 0.346 18.6 1E+05 0.379 16.8 1E+060.564 14.5 LA2 T7 provider (SEQ ID NO: 2) 0 0.091 28.2 SensitivityBlocker (SEQ ID NO: 29) 1E+02 0.311 29.8 @ 1E+02 Torch (SEQ ID NO: 50)1E+03 0.335 25.8 copies Non-T7 primer (SEQ ID NO: 22) 1E+04 0.331 22.9w/better 1E+05 0.336 20.4 separation 1E+06 0.476 18.2 Combo A T7provider (SEQ ID NO: 2) 0 0.024 Lower RFU Blocker (SEQ ID NO: 29) 1E+030.218 31.8 & later Torch (SEQ ID NO: 56) 1E+06 0.303 21.6 TTime @ 0Non-T7 primer (SEQ ID NO: 22) copies Combo B T7 provider (SEQ ID NO: 2)0 0.020 37.0 Lower RFU Blocker (SEQ ID NO: 29) 1E+03 0.243 32.6 & laterTorch (SEQ ID NO: 51) 1E+06 0.323 22.2 TTime @ 0 Non-T7 primer (SEQ IDNO: 22) copies Combo C T7 provider (SEQ ID NO: 2) 0 0.025 RFUs tooBlocker (SEQ ID NO: 29) 1E+03 0.150 30.8 low @ Torch (SEQ ID NO: 49)1E+06 0.205 21.1 other copy Non-T7 primer (SEQ ID NO: 22) levels Combo DT7 provider (SEQ ID NO: 2) 0 0.128 34.9 Blocker (SEQ ID NO: 29) 1E+030.176 30.8 Torch (SEQ ID NO: 50) 1E+06 0.332 20.3 Non-T7 primer (SEQ IDNO: 22) Combo E T7 provider (SEQ ID NO: 2) 0 0.206 35.1 Blocker (SEQ IDNO: 29) 1E+03 0.312 31.9 Torch (SEQ ID NO: 47) 1E+06 0.328 20.7 Non-T7primer (SEQ ID NO: 22) Combo F T7 provider (SEQ ID NO: 2) 0 0.119 36.5Blocker (SEQ ID NO: 29) 1E+03 0.391 33.2 Torch (SEQ ID NO: 47) 1E+060.486 21.9 Non-T7 primer (SEQ ID NO: 22) Combo G T7 provider (SEQ ID NO:2) 0 0.250 30.6 Blocker (SEQ ID NO: 29) 1E+03 0.504 30.4 Torch (SEQ IDNO: 57) 1E+06 0.664 19.8 Non-T7 primer (SEQ ID NO: 22) Combo H T7provider (SEQ ID NO: 2) 0 0.023 37.9 Lower RFU Blocker (SEQ ID NO: 29)1E+03 0.294 33.4 & later Torch (SEQ ID NO: 52) 1E+06 0.490 23.1 TTime @0 Non-T7 primer (SEQ ID NO: 22) copies

Table 13 summarizes the results from testing different torches forcross-reactions with related organisms. Specificity testing wasperformed using cell lysates of related organisms Pseudomonasaeruginosa, Pseudomonas putida, Myroides sp, Pseudomonas cepacia,Pseudomonas fluorescens, and Pseudomonas pickettii at ˜1E+05 colonyforming units (CFU), which is ˜1E+08 copies of rRNA.

TABLE 13 Summary of Torches Tested to Check for Cross-Reactions AveOligo Oligo CFU @ Range TTime Set Description (ID) 1E+05 (RFU) (min)Comments LA2 T7 provider (SEQ ID NO: 2) P. aeruginosa 0.206 15.7 CrossBlocker (SEQ ID NO: 29) P. putida 0.235 13.6 reaction w/3 Torch (SEQ IDNO: 50) Myroides sp. 0.189 12.5 organisms Primer (SEQ ID NO: 22) P.cepacia 0.051 30.2 P. fluorescens 0.231 17.7 P. pickettii 0.037 0.023LA2 T7 provider (SEQ ID NO: 2) P. aeruginosa 0.236 18.1 Cross amp w/Blocker (SEQ ID NO: 29) P. putida 0.090 21.6 reaction w/2 F11 Torch (SEQID NO: 51) Myroides sp. 0.118 20.5 organisms Torch Primer (SEQ ID NO:22) P. cepacia 0.033 P. fluorescens 0.089 24.1 P. pickettii 0.026 0.036

Example 5 Evaluation of Specific vs. Non-Specific Target Capture

Specific target capture (TC) oligonucleotides, SEQ ID NO:66-71, andspecific TC helper oligonucleotides, SEQ ID NO:72 & 73, were designed totarget Pseudomonas aeruginosa nucleic acid. A non-specific captureoligonucleotide, SEQ ID NO:65, having a random 2′-methoxy poly-(k)sequence with a poly-dT₃dA₃₀ tail, (k)₁₈-dT₃dA₃₀, where “k” is a randomassortment of guanine (G) and uracil (U) or thymine (T) basesincorporated into the oligonucleotide, was also synthesized.

TABLE 14 Target Capture Oligonucleotides SEQ Oligo ID Description NO:Sequence (5′-3′) Non-Specific 65 (k)₁₈ Target CaptureTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target Capture 66GCTCCTCTACCGCGTCACTTACGTGACACC TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATarget Capture 67 ccgcgucacuuacgugacaccTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target Capture 68 cuaccgcgucacuuacg-TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target Capture 69gcuccucuaccgcgucacuuacg- TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATarget Capture  70 CCCATTGTCGTTACTCATGTCAGCATTCGCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target Capture 71GCTTTTCACACCCATTGTCGTTACTCATGTCAGC TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATC-Helper 72 cgcagcuucggugugugguuugagc X TC Helper 73cucaacucaccuucacaggcuuacagaac X

The specific target capture oligos and helper oligos for Pseudomonasaeruginosa nucleic acid and were compared to non-specific targetcapture. Table 15 shows a summary of exemplary results obtained usingspecific target capture and helper oligos versus non-specific targetcapture oligo.

TABLE 15 Summary Table of Specific vs. Non-Specific Target CaptureOligos Oligo Set/ Copies Pae Ave Range TTime Com- Description (ID) rRNA(RFU) (min) ments Non-specific TC 0 0.053 6.7 (SEQ ID NO: 65) 1E+040.188 21.4 TC (SEQ ID NO: 69) 0 0.060 8.8 Helper (SEQ ID NO: 73) 1E+040.173 20.2 TC (SEQ ID NO: 68) 0 0.061 6.1 1E+04 0.166 20.4 TC (SEQ IDNO: 68) 0 0.064 8.4 Helper (SEQ ID NO: 73) 1E+04 0.160 21.3

Table 16 shows a summary of results for specific versus non-specifictarget capture in the presence of P. putida. These experimentsdemonstrated equivalence in performance and specificity between specifictarget capture oligos and non-specific target capture oligos whentesting the capture, amplification and detection of P. aeruginosanucleic acid by itself and in the presence of P. putida nucleic acid.

TABLE 16 Summary Table of Specific vs. Non-Specific Target CaptureOligos in the Presence of P. putida TC Oligo Set/ Copies Pae Ave RangeTTime Com- Description (ID) rRNA (RFU) (min) ments Non-specific TC 00.057 9.0 (SEQ ID NO: 65) 1E+03 0.128 20.9 1E+04 0.181 22.0 1E+P.putida0.178 21.6 TC (SEQ ID NO: 69) 0 0.059 4.7 Helper (SEQ ID NO: 73) 1E+030.119 19.1 1E+04 0.175 20.5 1E+P.putida 0.164 20.4 TC (SEQ ID NO: 68) 00.062 7.7 1E+03 0.095 16.9 1E+04 0.157 20.7 1E+P.putida 0.154 19.9 TC(SEQ ID NO: 68) 0 0.070 8.6 Helper (SEQ ID NO: 73) 1E+03 0.091 6.4 1E+040.142 19.5 1E+P.putida 0.141 20.4

Example 6 Reducing Cross-Reactivity with Organisms Related toPseudomonas Aeruginosa

This Example describes additional experiments performed in an effort toreduce cross-reactivity and amplification of organisms closely relatedto Pseudomonas aeruginosa. An exemplary graphical presentation ofreal-time assay data showing the signal obtained for 10 CFUs of P.aeruginosa as compared to 10⁵ CFU levels of closely relatedpseudomonads, P. stutzeri, P. pseudoalcaligenes, and P. mendocina, isshown in FIG. 1.

The AveRange (RFU) and TTime (min) or Cycle Time (min) results arepresented in tables below, as opposed to graphic representations. Aspreadsheet was created that analyzes the raw data file, and determinespositive and negative P. aeruginosa results along with assay validity. Apositive result is determined using a cutoff value of 750, a relativefluorescence unit (RFU) for the Trimmean cycles 71-76 from thebackground subtracted data. The validity of the assay, based on the UIC(internal control) results, is only confirmed for negative P. aeruginosaresults

The information provided in Table 17, is a subset of the experimentscompleted. Table 17 represents testing of lysates of these nearestneighbor Pseudomonas organisms, along with some environmental. Allexperimental results presented used the oligo set shown in Table 17. Onecolony forming unit (CFU) corresponds to 1000 copies of rRNA. The sameATCC strains identified as three cross-reacting organisms were testedand the results confirmed. Interestingly, when analyzing the graphicalrepresentations of these experiments (not shown), amplification with thecross-reacting organisms displayed plateaus.

TABLE 17 Check for Cross-Reactions with P. aeruginosa Real Time RTMAOligo ~1E+05 Re- Val- Com- Description (ID): copies RNA sults idityments Torch P. aeruginosa P 6/6 Cross (SEQ ID NO: 54) reaction w/3 T7provider P. stutzeri P 3/6 organisms (SEQ ID NO: 2) Blocker P.pseudoalcaligenes P 5/6 (SEQ ID NO: 29) Primer P. mendocina P 6/6 (SEQID NO: 24) Extender P. spinosa N 6/6 (SEQ ID NO: 44) Target Capture S.maltophila N 6/6 (SEQ ID NO: 69) TC Helper B. cepacia N 6/6 (SEQ ID NO:73) R. pickettii N 6/6 B. pyrocinia N 6/6 Negative Control N 6/6

Based on the results from Table 17, the next experiment with the threeorganisms looked at a titration of CFUs to determine if the samelow-level plateau occurs at the different levels.

Table 18 shows the results of the CFU titration as analyzed. Thetitration shows the same low-level amplification and plateau with onlythe emergence time varying based on the CFU level (not shown).

TABLE 18 Titration of Cross-Reacting Organisms with P. aeruginosa RealTime RTMA 1E+04 ~1E+02 ~1E+03 ~1E+04 ~1E+05 copies Organism CFU CFU CFUCFU RNA Validity Comments P. aeruginosa n/a n/a n/a n/a P 6/6 Cross P.stutzeri P/N P P P n/a 11/12 reaction P. pseudoalcaligenes P/N P P P n/a11/12 w/3 P. mendocina P/N P P/N P n/a 12/12 organisms Negative Controln/a n/a n/a n/a n/a 6/6 at all levels

The next experiment (Table 19) focused on assessing the Pseudomonasaeruginosa result in the presence of one of these cross-reactingorganisms. In previous specificity experiments, P. putida cross reactedwith a different oligo set than the final one chosen, so it was used asan additional control to look at Pseudomonas aeruginosa recovery. Theresults show the Pseudomonas aeruginosa signal is partially suppressedas the CFU level of P. putida increases (not shown), but greaterinhibition of Pseudomonas aeruginosa was seen with P. mendocina, whichwas not expected. The Pos/Neg results for P. putida in Table 19 werelow-level positives and not inconsistent when comparing replicates fromthe same target capture reaction.

TABLE 19 Titration of Cross-Reacting Organisms and P. aeruginosaRecovery 1E+04 ~1E+02 ~1E+03 ~1E+04 ~1E+05 copies Organism CFU CFU CFUCFU RNA Validity Comments P. aeruginosa (Pae) n/a n/a n/a n/a P 12/12 P.putida P/N N P/N N n/a 23/24 Suppression P. putida + Pae P P P P n/a24/24 of Pae signal P. mendocina P P P P n/a 24/24 Inhibition of P.mendocina + Pae P P P P n/a 24/24 Pae signal Negative Control n/a n/an/a n/a n/a 12/12

Based on sequence comparison of the P. mendocina with Pae and P. putida,complete inhibition of Pseudomonas aeruginosa amplification should nothave occurred, so the experiment was repeated with all three crossreacting organisms and analyzed with the TTime algorithm (Table 20). Asexpected, Pseudomonas aeruginosa amplification does occur in thepresence of these other organisms as opposed to the first result, withthe Pseudomonas aeruginosa RFU signal only being partially suppressed.

TABLE 20 Cross-Reacting Organisms and P. aeruginosa Recovery Ave ~CopiesRange TTime Organism RNA (RFU) (min) Comments P. aeruginosa (Pae) 00.077 64.3 P. aeruginosa 1E+06 0.873 15.8 P. stutzeri 1E+06 0.127 15.1Low level cross reaction P. stutzeri + Pae 1E+06 0.504 15.3 Suppressionof Pae signal P. pseudoalcaligenes 1E+06 0.121 13.5 Low level crossreaction P. pseudoalcaligenes + 1E+06 0.385 14.8 Suppression of Pae Paesignal P. mendocina 1E+06 0.113 17.6 Low level cross reaction P.mendocina + 1E+06 0.792 15.3 Suppression of Pae Pae signal

The results shown were tested using cell lysates with the exception ofPseudomonas aeruginosa, which was done using rRNA. Based on theunexpected results with these lysates, purified RNA from the organismswas used to do a similar Pseudomonas aeruginosa recovery experiment(Table 21). The same cross-reactions were seen with P. stutzeri, P.pseudoalcaligenes and P. mendocina, however there is no suppression ofthe Pseudomonas aeruginosa signal in the presence of these organism'srRNA at these levels.

TABLE 21 Cross-Reacting Organisms and P. aeruginosa Recovery ~CopiesAveRange TTime Com- Organism RNA (RFU) (min) ments P. aeruginosa (Pae) 00.052 P. aeruginosa 1E+04 0.400 24.4 P. aeruginosa 1E+06 0.485 19.1 P.stutzeri 1E+06 0.073 25.0 Low level cross reaction P. stutzeri + Pae1E+04 0.359 22.4 P. stutzeri + Pae 1E+06 0.439 17.9 P. pseudoalcaligenes1E+06 0.079 24.2 Low level cross reaction P. pseudoalcaligenes + 1E+040.370 22.5 Pae P. pseudoalcaligenes + 1E+06 0.497 17.6 Pae P. mendocina1E+06 0.084 24.7 Low level cross reaction P. mendocina + Pae 1E+04 0.40022.8 P. mendocina + Pae 1E+06 0.471 17.6 P. aeruginosa (TC) 0 0.058 11.7Incorrect TTime value P. aeruginosa (TC) 1E+04 0.143 25.6 P. aeruginosa(TC) 1E+06 0.249 21.8 P. stutzeri (TC) 1E+06 0.075 19.7 P. stutzeri +Pae (TC) 1E+04 0.149 24.7 P. stutzeri + Pae (TC) 1E+06 0.243 21.6 P.pseudoalcaligenes (TC) 1E+06 0.072 21.9 P. pseudoalcaligenes + 1E+040.150 25.1 Pae (TC) P. pseudoalcaligenes + 1E+06 0.262 21.6 Pae (TC) P.mendocina (TC) 1E+06 0.069 17.7 P. mendocina + Pae (TC) 1E+04 0.174 26.0P. mendocina + Pae (TC) 1E+06 0.257 23.0

These results indicate that the species-specific detection ofPseudomonas aeruginosa can be achieved by the present invention even inthe presence of closely related organisms, based upon thecharacteristics of the real-time TMA data (e.g., the size and shape ofRFU curves generated from the real-time TMA reactions). It is thereforepossible to use the methods of the present invention to not only detectthe presence of Pseudomonas aeruginosa, but also to detect other relatedorganisms that may be present. For example, if the observed RFU value isabove a suitable threshold, it could be concluded that the samplecontains Pseudomonas aeruginosa, and if lower RFU values are observedwithin a range determined to be indicative of the presence of otherclosely related organisms, then it can be conclude that the sample alsocontains one or more of these closely related organisms. Thus, themethods of the invention may be used in the simultaneous/differentialdetection of Pseudomonas aeruginosa and one or more of these relatedorganisms, based upon the distinct TMA signal characteristics exhibitedby the organisms.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A composition for use in a Pseudomonas aeruginosa nucleic acidamplification assay comprising a T7 provider oligo and a non-T7 oligo,wherein the T7 provider oligo targets a sequence in a region ofPseudomonas aeruginosa nucleic acid corresponding to bases from about725-825 of E. coli 23s rRNA and the non-T7 oligo targets the complementof a sequence in a region of Pseudomonas aeruginosa nucleic acidcorresponding to bases from about 845-950 of E. coli 23s rRNA.
 2. Thecomposition of claim 1, where the T7 provider targets a sequence in aregion of Pseudomonas aeruginosa nucleic acid corresponding to bases725-775 of E. coli 23s rRNA and the non-T7 primer targets the complementof a sequence in a region of Pseudomonas aeruginosa nucleic acidcorresponding to bases 900-950 of E. coli 23s rRNA.
 3. The compositionof claim 1, where the T7 provider targets a sequence in a region ofPseudomonas aeruginosa nucleic acid corresponding to bases 739-766 of E.coli 23s rRNA and the non-T7 primer targets the complement of a sequencein a region of Pseudomonas aeruginosa nucleic acid corresponding tobases 918-943 of E. coli 23s rRNA.
 4. The composition of claim 1, wherethe T7 provider is selected from SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:11or SEQ ID NO:14, and the non-T7 primer is selected from SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:19 or SEQ ID NO:15.
 5. The composition of claim1, where the T7 provider is SEQ ID NO:2 and the non-T7 primer is-SEQ IDNO:24.
 6. The composition of claim 1, further comprising a detectionoligo.
 7. The composition of claim 6, where the detection oligo is atorch oligo or molecular beacon.
 8. The composition of claim 7, wherethe torch oligo is selected from SEQ ID NO:51, SEQ ID NO:54, SEQ IDNO:50 or SEQ ID NO:56.
 9. The composition of claim 1, further comprisingan extend oligo.
 10. The composition of claim 9, where the extend oligois selected from SEQ ID NO:43 or SEQ ID NO:44.
 11. The composition ofclaim 1, further comprising a blocker oligo.
 12. The composition ofclaim 11, wherein the blocker oligo is selected from SEQ ID NO:29, SEQID NO:26, SEQ ID NO:40 or SEQ ID NO:42.
 13. A composition for use in aPseudomonas aeruginosa nucleic acid amplification assay comprising theT7 provider oligo, SEQ ID NO:2 and the non-T7 oligo, SEQ ID NO:24, andoptionally, further comprising the blocker oligo SEQ ID NO:29, the torcholigo SEQ ID NO:54, the extend oligo SEQ ID NO:44, the target captureoligo SEQ ID NO:69, and optionally, the target capture helper oligo SEQID NO:73.
 14. A kit for use in a Pseudomonas aeruginosa amplificationassay comprising a T7 provider oligo and a non-T7 oligo, wherein the T7provider oligo targets a sequence in a region of Pseudomonas aeruginosanucleic acid corresponding to bases from about 725-825 of E. coli 23srRNA, and the non-T7 oligo is targets the complement of a sequence in aregion of Pseudomonas aeruginosa nucleic acid corresponding to basesfrom about 845-950 of E. coli 23s rRNA.
 15. The kit of claim 14, wherethe T7 provider targets a sequence in a region of Pseudomonas aeruginosanucleic acid corresponding to bases 725-775 of E. coli 23s rRNA, and thenon-T7 primer targets the complement of a sequence in a region ofPseudomonas aeruginosa nucleic acid corresponding to bases 900-950 of E.coli 23s rRNA.
 16. The kit of claim 14, where the T7 provider targets asequence in a region of Pseudomonas aeruginosa nucleic acidcorresponding to bases 739-766 of E. coli 23s rRNA, and the non-T7primer targets the complement of a sequence in a region of Pseudomonasaeruginosa nucleic acid corresponding to bases 918-943 of E. coli 23srRNA.
 17. The kit of claim 14, where the T7 provider is selected fromSEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:11 or SEQ ID NO:14, and the non-T7primer is selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:19 or SEQID NO:15.
 18. The kit of claim 14, where the T7 provider is SEQ ID NO:2and the non-T7 primer is SEQ ID NO:24.
 19. The kit of claim 14, furthercomprising a detection oligo.
 20. The kit of claim 19, where thedetection oligo is a torch oligo or molecular beacon.
 21. The kit ofclaim 20, where the torch oligo is selected from SEQ ID NO:51, SEQ IDNO:54, SEQ ID NO:50 or SEQ ID NO:56.
 22. The kit of claim 14, furthercomprising an extend oligo.
 23. The kit of claim 22, where the extendoligo is selected from SEQ ID NO:43 or SEQ ID NO:44.
 24. The kit ofclaim 14, further comprising a blocker oligo.
 25. The kit of claim 24,wherein the blocker oligo is selected from SEQ ID NO:29 SEQ ID NO:26,SEQ ID NO:40 or SEQ ID NO:42.
 26. A kit for use in a Pseudomonasaeruginosa nucleic acid amplification assay comprising the T7 provideroligo, SEQ ID NO:2 and the non-T7 oligo, SEQ ID NO:24, and optionallyfurther comprising the blocker oligo SEQ ID NO:29, the torch oligo SEQID NO:54, the extend oligo SEQ ID NO:44, the target capture oligo SEQ IDNO:69 and, optionally, the target capture helper oligo SEQ ID NO:73. 27.A method for detecting the presence of Pseudomonas aeruginosa in asample, said method comprising performing a nucleic acid amplificationassay using a T7 provider oligo and a non-T7 oligo, wherein the T7provider oligo targets a sequence in a region of Pseudomonas aeruginosanucleic acid corresponding to bases from about 725-825 of E. coli 23srRNA, and the non-T7 oligo targets the complement of a sequence in aregion of Pseudomonas aeruginosa nucleic acid corresponding to basesfrom about 845-950 of E. coli 23s rRNA.
 28. The method of claim 27,where the T7 provider targets a sequence in a region of Pseudomonasaeruginosa nucleic acid corresponding to bases 725-775 of E. coli 23srRNA, and the non-T7 primer targets the complement of a sequence in aregion of Pseudomonas aeruginosa nucleic acid corresponding to bases900-950 of E. coli 23s rRNA.
 29. The method of claim 27, where the T7provider targets a sequence in a region of Pseudomonas aeruginosanucleic acid corresponding to bases 739-766 of E. coli 23s rRNA, and thenon-T7 primer targets the complement of a sequence in a region ofPseudomonas aeruginosa nucleic acid corresponding to bases 918-943 of E.coli 23s rRNA.
 30. The method of claim 27, where the T7 provider isselected from SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:11 or SEQ ID NO:14,and the non-T7 primer is selected from SEQ ID NO:22, SEQ ID NO:24, SEQID NO:19 or SEQ ID NO:15.
 31. The method of claim 27, where the T7provider is SEQ ID NO:2 and the non-T7 primer is SEQ ID NO:24.
 32. Themethod of claim 28, further comprising detecting amplified nucleic acidwith a detection oligo.
 33. The method of claim 32, where the detectionoligo is a torch oligo or molecular beacon.
 34. The method of claim 33,where the torch oligo is selected from SEQ ID NO:51, SEQ ID NO:54, SEQID NO:50 or SEQ ID NO:56.
 35. The method of claim 27, further comprisingan extend oligo.
 36. The method of claim 35, where the extend oligo isselected from SEQ ID NO:43or SEQ ID NO:44.
 37. The method of claim 28,further comprising a blocker oligo.
 38. The method of claim 37, whereinthe blocker oligo is selected from SEQ ID NO:29, SEQ ID NO:26, SEQ IDNO:40 or SEQ ID NO:42.
 39. The method of claim 27, where the T7 provideroligo is SEQ ID NO:2 and the non-T7 oligo is SEQ ID NO:24, and where themethod optionally includes the use of the blocker oligo SEQ ID NO:29,the torch oligo SEQ ID NO:54, the extend oligo SEQ ID NO:44, the targetcapture oligo SEQ ID NO:69 and, optionally, the target capture helperoligo, SEQ ID NO:73.
 40. The method of claim 33 wherein detecting saidamplified nucleic acid occurs in real-time.
 41. The method of claim 40wherein P. aeruginosa nucleic acid is specifically detected in thepresence of closely related Pseudomonads.
 42. The method of claim 40wherein the detection cut-off is set whereby P. aeruginosa nucleic acidand one or more closely related but not all Pseudomonads are detected.43. A composition for detecting Pseudomonas aeruginosa in a samplecomprising two or more amplification oligonucleotides designed toamplify the 800 region or the 1500 region of Pseudomonas aeruginosa 23sribosomal nucleic acid.
 44. The composition of claim 43, furthercomprising one or more probes for detecting Pseudomonas aeruginosa 23sribosomal nucleic acid.
 45. A method for detecting the presence ofPseudomonas aeruginosa in a sample, said method comprising performing anucleic acid amplification assay using the amplificationoligonucleotides of claim 43, and detecting amplified nucleic acid withone or more probes of claim 44.